Endothelial Cell Barrier Research Articles

P1296 Exploring the role of BOC mutations in Crohn’s disease using whole exome sequencing and molecular dynamics simulation

Abstract Background Crohn’s disease (CD) is a chronic inflammatory bowel disease, whose pathogenesis involves genetic component. In this study, BOC was identified as a potential risk gene for CD, whose gene product is a member of the cell adhesion molecule family. It is suggested that BOC binds to the N-terminus of Dhh protein (DhhN) through its Fn2-Fn3 domain, facilitating hh protein signal transduction. This signal has been shown to maintain endothelial cell barrier integrity, which is critical in the development of CD. Hence, we hypothesize that mutations in the BOC protein could alter its structure, impairing its ability to bind or release hh proteins, leading to abnormal hh signal transduction and affecting endothelial cell barrier function. Therefore, we employed molecular dynamics simulation to investigate the characteristics and affinity changes of the domain, and the domain with DhhN complex, both before and after mutation. Methods The target risk gene BOC was identified, and employed molecular dynamics simulation to investigate the characteristics and affinity changes of the BOC Fn2-Fn3 domain, and the domain with DhhN complex, both before and after mutation. Results 1. From the list of gene mutations, 121 candidate risk genes were identified. 2. The target risk gene BOC was identified, which is highly expressed on the colorectal endothelial cell membrane surface, and its expression shows a difference between CD patients and normal colorectal tissues. 3. The Arg697 mutation leads to decreased flexibility and increased stability of the BOC Fn2-Fn3 domain. 4. The Arg697 mutation increases the affinity of the domain with the DhhN complex, adding to the number of key amino acids and hydrogen bonds maintaining the complex’s affinity. Conclusion 1. Whole exome sequencing analysis revealed specific BOC gene mutations in CD patients, with differential expression between patients and healthy individuals suggesting an important role for BOC in CD. 2. Molecular dynamics simulations revealed that the Arg697 mutation increases the stability of BOC, and also showed increased stability and affinity of its binding with DhhN, which might prevent BOC from properly releasing hh proteins after binding, leading to abnormal hh signal transduction and thus impairing endothelial cell barrier function, which may promote the development and progression of CD. 3. The increased affinity of the complex following the Arg697 mutation is due to an increase in the number of key amino acid residues at the binding interface, resulting in more hydrogen bonds. Modifying these key amino acids to alter the affinity of the complex could normalize hh signal transduction, thereby restoring endothelial cell barrier function.

Trimetazidine: Activating AMPK Signal to Ameliorate Coronary Microcirculation Dysfunction after Myocardial Infarction.

Myocardial ischemia-reperfusion (I/R) injury and coronary microcirculation dysfunction (CMD) are observed in patients with myocardial infarction after vascular recanalization. The antianginal drug trimetazidine has been demonstrated to exert a protective effect in myocardial ischemia-reperfusion injury. This study aimed to investigate the role of trimetazidine in endothelial cell dysfunction caused by myocardial I/R injury and thus improve coronary microcirculation. The myocardial I/R mouse model was established, and trimetazidine was administered for 7 days before myocardial I/R model establishment. Echocardiography, 2,3,5-triphenyltetrazolium chloride (TTC) staining, hematoxylin-eosin (H&E) staining, and thioflavin S staining were applied to assess myocardial injury and microvascular function. Additionally, the oxygen-glucose deprivation/reperfusion (OGD/R) model was developed in endothelial cells to simulate myocardial I/R injury in vitro. Griess reaction method, immunofluorescence, and western blotting (WB) were employed to detect the expressions of nitric oxide (NO), platelet endothelial cell adhesion molecule-1 (CD31) and vascular endothelial (VE)-cadherin, zonula occludens protein 1 (ZO-1), occludin, vascular endothelial growth factor (VEGF) and adenosine monophosphate (AMP)-activated protein kinase (AMPK) signaling-related proteins in endothelial cells and mouse cardiomyocytes. AMPK pathway inhibitor compound C was used for further mechanism validation. Our research demonstrated that trimetazidine can alleviate myocardial pathological injury and cardiac function injury during myocardial I/R. Trimetazidine was observed to improve microvascular reflux phenomenon and microvascular function and barrier injury in myocardial I/R and OGD/R models. Additionally, the expressions of AMPK signal-related proteins were found to be inhibited in myocardial I/R and OGD/R models, which were then activated in mice administered trimetazidine. However, the effects of trimetazidine on endothelial cell function and barrier damage were attenuated after co-treatment with compound C and trimetazidine. Trimetazidine ameliorated myocardial I/R-induced CMD by activating AMPK signaling.

Inhibition of platelet activation alleviates diabetes-associated cognitive dysfunction via attenuating blood-brain barrier injury.

Cognitive dysfunction has become the second leading cause of death among the diabetic patients. In pre-diabetic stage, blood-brain barrier (BBB) injury occurs and induced the microvascular complications of diabetes, especially, diabetes-associated cognitive dysfunction (DACD). Endothelial cells are the major component of BBB, on which the increased expression of CD40 could mediate BBB dysfunction in diabetics. Since platelets play an important role in regulating endothelial cell barrier function and over 95 % of the circulating soluble CD40 ligand (sCD40L) is derived from activated platelets, we speculated that the release of CD40L from activated platelets induced by diabetes was the key mechanism that aggravated BBB injury and leaded to DACD. We performed inhibition of platelet activation on diabetic and non-diabetic mice, with or without cilostazol treatment, and then compared cognitive function, platelet activation, BBB structure and permeability. In vitro, mouse brain microvascular endothelial cell line (b.End3) were exposed to CD40L for 24 h at 5.5 mM or 30 mM glucose media after silencing CD40 and HIF1α or not to investigate the effects of CD40 on BBB disruption and the underlying molecular pathways. Inhibition of platelet activation improved cognitive behaviors in diabetic mice, accompanied with reduced BBB permeability, increased tight junction proteins, balanced Aβ transporters, as well as attenuated Aβ deposition and hippocampal neurons damage. In vitro, CD40L increased HIF1α, diminished tight junction proteins and dysregulated Aβ transporters in b.End3 cells, which could be restored by CD40 siRNA and HIF1α siRNA. Hence, inhibition of platelet activation ameliorates DACD via alleviating BBB injury, which involving the regulation of CD40L-CD40-HIF1α signaling pathway. Our study may demonstrate a potential therapeutic target for the treatment of DACD.

Protection of liver sinusoidal endothelial cells using different preservation solutions.

Donor liver preservation methods and solutions have evolved over the last years. Liver sinusoidal endothelial cell (LSEC) barrier function and integrity during preservation is crucial for outcomes of liver transplantation. Therefore, the present study aimed to determine optimal preservation of LSEC barrier function and integrity, using different preservation solutions. Human Umbilical Vein Endothelial Cells (HUVEC) and LSEC were incubated in either University of Wisconsin machine perfusion solution (UW-MPS), histidine-tryptophan-ketoglutarate (HTK), or endothelial cell growth medium 2 (EGM2) (as a golden standard for cell culturing). Endothelial integrity was assessed by measurement of cellular morphology and expression of membrane proteins ; PECAM-1, ICAM-1, and Fc-gamma receptor CD32b (FcΥRCD32b). Endothelial barrier function was measured by electric cell-substrate impedance sensing (ECIS). Cellular respondence to inflammatory stimuli with tumor necrosis factor-alpha (TNF-α), was tested by studying trans-endothelial migration (TEM) under flow conditions. Differences in these parameters were analyzed between the different preservation solutions. PECAM-1 expression was high for all preservation solutions in HUVEC and LSEC. ICAM-1 expression was increased in both LSEC and HUVEC in all preservation solutions plus TNF-α. UW reduced PECAM-1 expression, whereas EGM2 medium promoted barrier function in LSEC and HUVEC, and monolayer recovery after wounding was best achieved in cells incubated in EGM2. LSEC and HUVEC incubated with EGM2 plus TNF-α both supported neutrophil adhesion and TEM, but much less to none when incubated in UW plus TNF-α. Overall, EGM2 showed the best results in preserving endothelial barrier function for both HUVEC and LSEC.

Impacts of APOE-ε4 and exercise training on brain microvascular endothelial cell barrier function and metabolism

The APOE-ε4 genotype is the highest genetic risk factor for Alzheimer's disease (AD), and exercise training can reduce the risk of AD. Two early pathologies of AD are degradation of tight junctions between brain microvascular endothelial cells (BMEC) and brain glucose hypometabolism. Therefore, the objective of this work was to determine how the APOE-ε4 genotype and serum from exercise trained individuals impacts BMEC barrier function and metabolism. iPSC homozygous for the APOE-ε3 and APOE-ε4 alleles were differentiated to BMEC-like cells and used to measure barrier function and metabolism. To investigate exercise effects, serum was collected from older adults pre- and post- 6 months of exercise training (n=9 participants per genotype). APOE-ε3 and APOE-ε4 BMEC were treated with genotype-matched serum, and then barrier function and metabolism were measured. APOE-ε4 genotype impaired BMEC barrier function and metabolism by reducing sirtuin 1 (SIRT1) levels by 27% (p=0.0188) and baseline insulin signalling by 37% (p=0.0186) compared to APOE-ε3 BMEC. Exercise-trained serum increased SIRT1 by 33% (p=0.0043) in APOE-ε3 BMEC but decreased SIRT1 by 22% (p=0.0004) in APOE ε4 BMEC. APOE-ε4 directly impairs glucose metabolism and barrier function. Serum from exercise trained individuals alters SIRT1 in a genotype-dependent manner but may require additional cues from exercise to decrease AD pathologies. Brain and Behaviour Initiative at the University of Maryland through the Seed Grant Program, NSF-GRFP DGE 1840340, Fischell Fellowship in Biomedical Engineering, NSF CBET-2211966 and DGE-1632976, National Niemann-Pick Disease Foundation, University of Maryland ASPIRE Program, NIH R01HL165193, R01HL140239-01, and R01AG057552.

FAK activity exacerbates disturbed flow-mediated atherosclerosis via VEGFR2-Cbl-NF-κB signaling.

Atherosclerosis develops at predictable sites in the vasculature where branch points and curvatures create non-laminar disturbed flow. This disturbed flow causes vascular inflammation by increased endothelial cell (EC) barrier permeability and the expression of inflammatory genes such as vascular cell adhesion molecule-1 (VCAM-1). Vascular endothelial growth factor receptor 2 (VEGFR2) is important for flow-induced EC inflammation; however, there are still some gaps in the signaling pathway. Focal adhesion kinase (FAK) is a protein tyrosine kinase whose expression has been implicated in flow-mediated signaling in ECs. However, the link between FAK and VEGFR2 in flow-mediated inflammation signaling has remained unelucidated. Here we found that priming of VEGFR2 with VEGF was critical for flow-mediated activation of FAK and NF-κB. Mechanistically, FAK activation triggers tyrosine phosphorylation of Casitas B-lineage lymphoma (CBL; an E3 ubiquitin ligase) that interacts with VEGFR2 under flow conditions. Further, Apoe-/- mice fed a western diet (WD) exhibited increased FAK activity within the atheroprone disturbed flow region of the inner aortic arch compared to the outer arch. Disturbed flow-induced FAK activation is associated with elevated VEGFR2 on the surface of ECs of the inner aortic arch, but not in the outer arch. Taken together, these data suggest that suppression of augmented FAK activity under disturbed flow may prove beneficial in reducing pro-inflammatory signaling of the endothelial layer.

Endothelial Pim3 kinase protects the vascular barrier during lung metastasis

Endothelial cells (ECs) form a tissue-specific barrier for disseminating cancer cells in distant organs. However, the molecular regulation of the ECs in the metastatic niche remains unclear. Here, we analyze using scRNA-Seq, the transcriptional reprogramming of lung ECs six hours after the arrival of melanoma cells in mouse lungs. We discover a reactive capillary EC cluster (rCap) that increases from general capillary ECs in response to infiltrating cancer cells. rCap is enriched for angiogenic and inflammatory pathways and is also found in human lung datasets. The JAK-STAT activated oncogenic Pim3 kinase is a marker of rCap, being upregulated in spontaneous metastasis models. Notably, PIM inhibition increases vascular leakage and metastatic colonization and impairs the EC barrier by decreasing the junctional cadherin-5 and catenins α, β and δ. These results highlight the pulmonary endothelium’s plasticity and its protection by PIM3, which may impair the efficacy of PIM inhibitors in cancer therapies.

On-chip fabrication of tailored 3D hydrogel scaffolds to model cancer cell invasion and interaction with endothelial cells.

The high mortality associated with certain cancers can be attributed to the invasive nature of the tumor cells. Yet, the complexity of studying invasion hinders our understanding of how the tumor spreads. This work presents a microengineered three-dimensional (3D) in vitro model for studying cancer cell invasion and interaction with endothelial cells. The model was generated by printing a biomimetic hydrogel scaffold directly on a chip using 2-photon polymerization that simulates the brain's extracellular matrix. The scaffold's geometry was specifically designed to facilitate the growth of a continuous layer of endothelial cells on one side, while also allowing for the introduction of tumor cells on the other side. This arrangement confines the cells spatially and enables in situ microscopy of the cancer cells as they invade the hydrogel scaffold and interact with the endothelial layer. We examined the impact of 3D printing parameters on the hydrogel's physical properties and used patient derived glioblastoma cells to study their effect on cell invasion. Notably, the tumor cells tended to infiltrate faster when an endothelial cell barrier was present. The potential for adjusting the hydrogel scaffold's properties, coupled with the capability for real-time observation of tumor-endothelial cell interactions, offers a platform for studying tumor invasion and tumor-endothelial cell interactions.

1,8-Cineole reduces pulmonary vascular remodelling in pulmonary arterial hypertension by restoring intercellular communication and inhibiting angiogenesis.

Pulmonary Arterial Hypertension (PAH) is characterized by pulmonary vascular remodelling, often associated with disruption of BMPR2/Smad1/5 and BMPR2/PPAR-γ signalling pathways that ultimately lead to right ventricle failure. Disruption of intercellular junctions and communication and a pro-angiogenic environment are also characteristic features of PAH. Although, current therapies improve pulmonary vascular tone, they fail to tackle other key pathological features that could prevent disease progression. In this scenario, aromatic plants emerge as promising sources of bioactive compounds, with 1,8-cineole standing out due to its hypotensive properties and cardioprotective effect in PAH. The present study aims to explore for the first time the effect of 1,8-cineole in pulmonary vascular remodelling associated with PAH. Resorting to the monocrotaline (MCT)-induced PAH animal model, the effect of 1,8-cineole on vascular remodelling including interstitial collagen accumulation, smooth muscle cell proliferation and protein levels of BMPR2 pathway-related proteins, was assessed by microscopy and western blot (WB) analysis. The integrity of gap junctions, pulmonary surfactant, mitochondrial structure and endothelial cell barrier were evaluated by transmission electron microscopy, confocal microscopy and WB analysis. Furthermore, the effect of 1,8-cineole on angiogenesis was determined on pulmonary artery endothelial cells (PAEC) submitted to hypoxia using the scratch wound and Matrigel angiogenesis assays, and the number of sprouts on isolated healthy and diseased pulmonary artery rings, treated with the compound, enabled the validation of these effects. 1,8-Cineole mitigated PAH-associated derailment of both BMPR2/Smad1/5 and BMPR2/PPAR-γ pathways and concomitantly reduced interstitial fibrosis and the arterial medial layer thickness in pulmonary arteries. The compound restored gap junction, lung surfactant and mitochondrial integrity and preserved endothelial barrier integrity. Furthermore, 1,8-cineole exerted an anti-angiogenic effect, by impairing the formation of vessel-like structures in PAEC and sprouting formation in isolated pulmonary arteries. The present study brings new insights about the mechanisms whereby 1,8-cineole impacts pulmonary vascular remodelling and demonstrates the potential of 1,8-cineole as a therapeutic strategy to hamper PAH progression.

Retraction: Hyperglycemia via activation of thromboxane A2 receptor impairs the integrity and function of blood-brain barrier in microvascular endothelial cells.

Retraction: Hyperglycemia via activation of thromboxane A2 receptor impairs the integrity and function of blood-brain barrier in microvascular endothelial cells.

Ixochymostatin, a trypsin inhibitor-like (TIL) protein from Ixodes scapularis, inhibits chymase and impairs vascular permeability

Ticks obtain a blood meal by lacerating small blood vessels and ingesting the blood that flows to the feeding site, which triggers various host responses. However, ticks face the challenge of wound healing, a process involving hemostasis, inflammation, cell proliferation and migration, and remodeling, hindering blood acquisition. To overcome these obstacles, tick salivary glands produce an array of bioactive molecules. Here, we characterize ixochymostatin, an Ixodes scapularis protein belonging to the trypsin inhibitor-like (TIL) family. It is expressed in multiple developmental stages and in tick salivary glands and acts as a slow and tight-binding inhibitor of chymase, cathepsin G, and chymotrypsin. Predictions for the tertiary structure complex between ixochymostatin and chymase suggest a direct interaction between the inhibitor reactive site loop and protease active sites. In vitro, ixochymostatin protects the endothelial cell barrier against chymase degrading action, decreasing cell permeability. In vivo, it reduces vascular permeability induced by chymase and compound 48/80, a mast cell degranulator agonist, in a mouse model. Additionally, ixochymostatin inhibits the chymase-dependent generation of vasoconstrictor peptides. Antibodies against ixochymostatin neutralize its inhibitory properties, with epitope mapping identifying potential neutralization regions. Ixochymostatin emerges as a novel tick protein modulating host responses against tick feeding, facilitating blood acquisition.

Abstract 4135700: Effects of Mitochondrial Dysfunction on Extracellular Vesicle Release and Subsequent Disruption of the Lung Vascular Endothelial Barrier

Background: Lung endothelial cell (EC) barrier disruption is a hallmark of acute lung injury (ALI), which is commonly caused by bacterial pneumonia. Alveolar epithelial cells (AEC) are the first line of defence against invading respiratory pathogens in the lungs and can mediate inflammatory signaling to the adjacent EC contributing to barrier disruption. We have previously demonstrated that pneumolysin (PLY), a major Streptococcus pneumoniae virulence factor, induces both mitochondrial dysfunction in lung AEC and release of large extracellular vesicles (EVs). These EVs, which are enriched with mitochondrial cargo, contribute to increased permeability of the lung EC barrier. Here, we investigate strategies to inhibit mitochondrial dysfunction in AEC and examine how these interventions impact EC barrier function. Methods: Human alveolar epithelial cells (A549) or immortalized AEC (Cell Biologics) were pre-treated with mitoTEMPO (mtROS scavenger), P110 (excessive fission inhibitor), or ensifentrine [dual phosphodiesterase (PDE) 3/4 inhibitor] prior to PLY treatment (100-300 ng/ml, 4 hrs). Mitochondrial function was assessed by the JC-1 assay. EVs were isolated from cell media and analyzed for their mitochondrial content (Tom20 and Tim23 expression). To assess their functional role, EVs were added to TNF-α treated human lung microvascular EC, and barrier function was then determined using the ECIS assay to measure transendothelial electrical resistance (TER). Results: PLY-induced mitochondrial dysfunction was reduced in the presence of mitoTempo or P110. EVs from PLY-treated AEC (EV PLY ; enriched mitochondrial content) exacerbated TNF-α-induced EC barrier permeability as indicated by the significant decrease in TER. EV PLY derived from MitoTEMPO or P110-pretreated AEC contained less mitochondrial cargo and resulted in less EC permeability after TNF-α. Compared to mitoTEMPO and P110, ensifentrine exhibited superior protection against mitochondrial dysfunction after PLY exposure. In addition, in the presence of ensifentrine, the quantity of EV release and their extracelllular mitochondrial content were both dramatically reduced. In contrast to control EV PLY , EV PLY from ensifentrine-pretreated AEC demonstrated no barrier disruptive properties. Conclusions: Mitochondrial dysfunction in AEC contributes to vascular endothelial injury in ALI through mechanisms involving the release of barrier-disruptive EVs, which can be effectively targeted by inhibiting PDE3/4.

Circulating mtNFPs Are Associated with ARDS after CPB and Regulate Endothelial Barrier through FPR2.

Cardiopulmonary bypass (CPB) increases the risk of acute respiratory distress syndrome (ARDS) due to endothelial cell (EC) barrier dysfunction. However, the specific role of mitochondrial N-formyl peptides (mtNFPs) in ARDS following CPB remains unexplored. Here, we investigated the differential expression of circulating mtNFPs in patients after CPB, focusing on the novel role of FPR2 in ECs. Levels of circulating mtNFPs were assessed using enzyme-linked immunosorbent assay (ELISA). Several mtNFPs (ND4, ND5, ND6, and Cox1) were significantly upregulated in patients with ARDS at day 1 post-CPB compared to patients without ARDS. Higher levels of ND6 were correlated with worst PaO2/FiO2 (r=-0.2219 and P<0.0001) and cardiac Troponin T (r=2.107 and P<0.0001). Utilizing patient-derived serum and a rat lung ischemia reperfusion injury (LIRI) model, we observed a positive correlation between serum ND6 concentration and ARDS, which is also associated with EC barrier dysfunction. In vitro experiments, using trans-endothelial electric resistance (TEER) measurements and fluorescence microscopy with FITC-labeled VE-cadherin, demonstrated that ND6 disrupts the EC barrier through FPR2. Furthermore, FPR2 controls the release of ND6 out of mitochondria and cytoplasm under hypoxia reoxygenation (HR). Activated FPR2 leads to upregulation of nuclear transcription factor-kappa B (NF-κB) by inducing IκBα phosphorylation, promoting ICAM1 and VCAM1 expression, thereby compromising EC barrier integrity. Circulating pro-inflammatory and barrier-disruptive mtNFPs, particularly ND6, are associated with ARDS in patients undergoing CPB. The novel ND6-FPR2 axis regulates inflammation and EC permeability through the NF-κB pathway.

MiR-144/451: A Regulatory Role in Inflammation.

Inflammation is the natural defense mechanism of the body in response to injury, infection, or other stimuli. Excessive or persistent inflammatory responses can lead to the development of inflammatory diseases. Therefore, elucidating the regulatory mechanisms of inflammatory cells is crucial for understanding the pathogenesis of such diseases and devising novel therapeutic approaches. Moreover, miR-144/451 plays an important role in erythroid maturity and tumour development. Herein, we have reviewed the regulatory role of miR-144/451 in inflammation. Papers on miR-144, miR-451, and inflammation were retrieved from PubMed and Web of Science to be analysed and summarised. miR-144/451 plays a significant role in modulating inflammatory responses. Pro- and anti-inflammatory gene transcription is regulated by miR-144/451 binding to the 3' untranslated regions. Studies have shown that miR-451 inhibits the activation of various inflammatory cells, including macrophages, neutrophils, and T lymphocytes, thereby reducing the release of inflammatory mediators. However, miR-144 expression varies in different inflammatory diseases. miR-144 expression is downregulated in macrophages after induction by lipopolysaccharide, cysteine, or Mycobacterium tuberculosis, which promotes the secretion of inflammatory mediators; nonetheless, miR-144-3p overexpression in macrophages can aggravate atherosclerosis. Meanwhile, miR-144 overexpression prevents disruption of the lung endothelial cell barrier, whereas it exacerbates endothelial cell injury in Crohn's disease. miR-144/451 may serve as a potential target for the treatment of inflammatory diseases.

Histidine containing dipeptides protect epithelial and endothelial cell barriers from methylglyoxal induced injury

Integrity of epithelial and endothelial cell barriers is of critical importance for health, barrier disruption is a hallmark of numerous diseases, of which many are driven by carbonyl stressors such as methylglyoxal (MG). Carnosine and anserine exert some MG-quenching activity, but the impact of these and of other histidine containing dipeptides on cell barrier integrity has not been explored in detail. In human proximal tubular (HK-2) and umbilical vein endothelial (HUVEC) cells, exposure to 200 µM MG decreased transepithelial resistance (TER), i.e. increased ionic permeability and permeability for 4-, 10- and 70-kDa dextran, membrane zonula occludens (ZO-1) abundance was reduced, methylglyoxal 5-hydro-5-methylimidazolones (MG-H1) formation was increased. Carnosine, balenine (ß-ala-1methyl-histidine) and anserine (ß-ala-3-methyl-histidine) ameliorated MG-induced reduction of TER in both cell types. Incubation with histidine, 1-/3-methylhistidine, but not with ß-alanine alone, restored TER, although to a lower extent than the corresponding dipeptides. Carnosine and anserine normalized transport and membrane ZO-1 abundance. Aminoguanidine, a well-described MG-quencher, did not mitigate MG-induced loss of TER. Our results show that the effects of the dipeptides on epithelial and endothelial resistance and junction function depend on the methylation status of histidine and are not exclusively explained by their quenching activity.

A DISEASE-BASED PERSPECTIVE OF THE RELATIONSHIP BETWEEN NEUROINFLAMMATION AND IMPAIRED GLUCOSE METABOLISM

Neuroinflammation is a significant contributor to the pathogenesis of several central nervous system disorders including Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, and amyotrophic lateral sclerosis. Neuroinflammation is the immune response of the central nervous system against central or peripheral abnormalities disturbed by foreign agents, molecules, metabolic activities, or various diseases. Astrocytes and microglia activation are the main activators of neuroinflammation. The polarization changes of these defender cells have some key roles in bodily metabolism as much as neuronal behavior. The blood-brain barrier is known as the first defender of brain parenchyma. Neuroinflammation disrupts blood-brain barrier integrity and may cause blood-brain barrier breakdown. Glucose is the main energy source of brain and glucose uptake is achieved through the blood-brain barrier. Altered glucose metabolism may have detrimental effects on brain functions and may cause brain disorders. Also, it has been suggested that neuroinflammation may have crucial roles in glucose metabolism. The distribution of the blood-brain barrier in vascular endothelial cells of neurons, astrocytes, and microglia contributes to the transport of glucose to the cells of brain. Microglia and astrocyte polarization are suggested as the two main underlying mechanisms in neuroinflammation. It’s obviously determined that neuroinflammation-caused neurodegenerative diseases are tightly linked with the brain insulin resistance and disrupted cerebral and peripheral glucose metabolism. However, there is lacking knowledge about glucose metabolism deficiencies and microglia/astrocyte polarization. Herein this review, we summarized the neuroinflammation and glucose metabolism with the most common neurological diseases and the possible effects of microglia/astrocyte polarization on glucose metabolism.

CAMP/PKA-mediated in vitro angiogenesis: A novel interplay between PKA, RhoA/ROCK, and VEGFR2 signalling

Abstract Background and Aims Others and we have previously demonstrated that cAMP/PKA signalling stabilise endothelial cell (EC) barrier function via modulating the activities of Rho GTPases, mainly RhoA and Rac1. In the present study we analysed whether cAMP/PKA also modulates EC angiogenesis capacity and whether Rho GTPases are involved in this function. Methods The study was carried out on cultured human umbilical vein ECs (HUVECs). RhoA and Rac1 activities were genetically manipulated by lentiviral-mediated over expression of dominant negative (DN) and constitutive active (CA) forms of RhoA and Rac1. Pharmacologically, specific activation and inhibition of PKA was achieved by using cAMP analogue N6-Benzoyl-cAMP (5 mM) and PKI (1 µM), respectively. Pharmacologically, Rac1 was inhibited using NSC 23766 (10 µM) and RhoA was activated using Rho activator I (Cytoskeleton Inc.). In vitro angiogenesis was analysed by 3-D spheroid assay and matrigel tube formation assay. Results Pharmacologically, specific activation of PKA induced EC migration (Wound healing assay), tube formation, and 3-D spheroids and potentiated the VEGF-induced in vitro angiogenesis. These pro-angiogenesis activities were abrogated by highly specific cell-permeable peptide inhibitor (PKI) of PKA. Activation of cAMP/PKA-signalling resulted in an increased VEGFR2 and Akt phosphorylation, increased Rac1 activity, and a strong inhibition of RhoA/Rock pathway. Pharmacological activation of RhoA but not inhibition of Rac1 abrogated PKA-induced VEGFR2 phosphorylation, tube formation and 3-D spheroids. Lentiviral mediated over-expression of constitutive active form of RhoA but not Rac1 abrogated PKA-induced VEGFR2 and Akt phosphorylation and angiogenesis. Moreover, pharmacological inhibition of Akt also abrogated PKA-mediated VEGFR2 phosphorylation and angiogenesis. Likewise, over expression of either dominant negative RhoA or pharmacological inhibitors of Rho kinase (ROCK) promoted basal and potentiated PKA-mediated VEGFR2 and Akt phosphorylation and angiogenesis. Conclusion We describe for the first time a novel interplay between PKA, RhoA/ROCK, Akt, and VEGFR2 signalling and angiogenesis. Inhibition of RhoA/ROCK plays an important role in mediating PKA-induced angiogenesis and activation of RhoA/ROCK signalling may offer an important target to intervene therapeutic angiogenesis.

APPLICATION OF IN VITRO MODELS: INCORPORATING BLOOD BRAIN BARRIER MODELS WITH GLIOMA, AND THE USE OF MICROflUIDICS

Abstract AIMS The blood brain barrier (BBB) presents one of the main obstacles for the development of novel glioblastoma treatments. The various transporters and enzymes within the BBB reduce the disposition of novel therapies into the brain. There is a need for an in vitro BBB-glioma model with proven efficacy of predicting CNS delivery at earlier stages in the drug discovery pipeline. METHOD A BBB-glioma lab-on-a-chip model could enable rapid identification of novel therapies that can cross the BBB at earlier stages of the drug development process, before going to clinical trials. The application of microfluidic technology allows for the detection of soluble tissue-derived factors and the assessment of the levels of soluble markers released from malignant tissue. Adding ‘flow’ to the model, is more reflective of the dynamic flow present at the BBB. RESULTS Validation studies of the current BBB model and converting this model over to a lab-on-a-chip model have shown the presence of an endothelial cell barrier and the tight junction proteins Zo-1 and Claudin-1, through western blotting, immunocytochemistry, and permeability experiments using varying molecular weights of FitC-dextran. These validation studies have been completed using brain endothelial monolayers on polycarbonate transwell inserts, with the immortalised cell line HCMEC/d3, and the short-term culture cells HBMEC. Also, validation studies using a co-culture of endothelial cells, astrocytes and pericytes have begun, using the same techniques to show a barrier has formed, on both the current BBB model and the lab-on-a-chip model. CONCLUSION The aim of this study is to combine the use of an all-human BBB-glioma model with lab-on-a-chip technology to produce a high throughput model to test novel therapies at an earlier stage. The lab-on-a-chip model will be validated for the purpose of measuring permeability and drug response and to investigate the expression of novel targets verses expression in conventional models.

Rapid precision targeting of nanoparticles to lung via caveolae pumping system in endothelium.

Modern medicine seeks precision targeting, imaging and therapy to maximize efficacy and avoid toxicities. Nanoparticles (NPs) have tremendous yet unmet clinical potential to carry and deliver imaging and therapeutic agents systemically with tissue precision. But their size contributes to rapid scavenging by the reticuloendothelial system and poor penetration of key endothelial cell (EC) barriers, limiting target tissue uptake, safety and efficacy. Here we discover the ability of the EC caveolae pumping system to outpace scavenging and deliver NPs rapidly and specifically into the lungs. Gold and dendritic NPs are conjugated to antibodies targeting caveolae of the lung microvascular endothelium. SPECT-CT imaging and biodistribution analyses reveal that rat lungs extract most of the intravenous dose within minutes to achieve precision lung imaging and targeting with high lung concentrations exceeding peak blood levels. These results reveal how much ECs can both limit and promote tissue penetration of NPs and the power and size-dependent limitations of the caveolae pumping system. This study provides a new retargeting paradigm for NPs to avoid reticuloendothelial system uptake and achieve rapid precision nanodelivery for future diagnostic and therapeutic applications.

Drug-Induced Mitochondrial Disruption in the Blood-Brain Barrier Cells: Overlooked Player in Drug Safety Evaluation.

The blood-brain barrier (BBB) is based on the unique pattern of the microvasculature of the central nervous system (CNS), which controls the transport of molecules between the CNS and the blood. The blood-brain barrier is mainly composed of endothelial cells, pericytes, and basement membrane, as well as the astrocytes and immune cells as perivascular macrophages and microglial cells. The dysfunction of this barrier can cause serious neuronal disorders due to the transport of hazardous molecules and immune cells to the CNS. Mitochondria plays a major role in cellular homeostasis in terms of health and disease. This review evaluated the published data about the effect of the drugs on the cells of BBB. Only seven articles were found that considered the effect of drugs on the barrier endothelial cells and mitochondria via different assays. Further studies are recommended to evaluate the impact of used medications on BBB cell bioenergetics. Also, the effect of the newly studied pharmaceutical agents on the BBB bioenergetics should be included within their safety profile studies.