Endothelial Cell Disruption Research Articles

Impact of cell culture parameters on production and vascularization bioactivity of mesenchymal stem cell-derived extracellular vesicles.

Mesenchymal stem cell (MSC)‐derived extracellular vesicles (EVs) have emerged as potential therapeutic agents for numerous applications. EVs offer potential advantages over cell‐based therapies with regard to safety, stability and clearance profiles, however production and potency limitations must be addressed to enable eventual translation of EV‐based approaches. Thus, we sought to examine the role of specific cell culture parameters on MSC EV production and bioactivity toward informing rational design parameters for scalable EV biomanufacturing. We report significantly reduced MSC EV vascularization bioactivity, as measured by an endothelial cell gap closure assay, with increasing passage in culture by trypsinization, especially beyond passage 4. We further show that increased frequency of EV collection yielded higher numbers of EVs from the same initial number of MSCs over a 24 hr period. Finally, we demonstrate that decreased cell seeding density in culture flasks resulted in increased production of EVs per cell in MSCs and other cell types. Overall, these studies highlight the need for careful consideration of the parameters of cell passage number and cell seeding density in the production of therapeutic EVs at laboratory scale and for rational design of large‐scale EV biomanufacturing schemes.

Curcumin-primed and curcumin-loaded exosomes: potential neural therapy.

Curcumin-primed and curcumin-loaded exosomes: potential neural therapy.

Posterior Reversible Encephalopathy Syndrome Triggerred By Alcohol Withdrawal

Posterior reversible encephalopathy syndrome (PRES) is a clinico-radiological entity characterized by headache, altered mental status, epileptic seizures, visual disturbances and typically transient changes in posterior cerebral circulation areas. In this article, we present a case of alcohol withdrawal accompanied by PRES. A 53-year-old male patient presented to the emergency department with visual hallucinations and meaningless speech. History from his relatives revealed that he has been consuming alcohol for about 35 years and the last consumption was 3 days before the admission. On neurological examination, there was limited cooperation and disorientation was evident to person, place and time. The speech was incoherent. No localizing sign was observed. Cranial magnetic resonance imaging (MRI) revealed bilateral hyperintense areas in medial occipital cortices and in subcortical white matter extending partly into parietal region. Treatment for alcohol withdrawal was started. Signs and symptoms regressed on the 7th day of the treatment as well as the lesions on MRI. The clinical presentation, characteristic MRI features together with the reversible nature of the syndrome suggest the diagnosis of PRES. The precise pathophysiological mechanism of PRES still remains unclear. Hypertension, clinical conditions that are associated with impaired cerebral auto-regulation as well as alcohol use which increases the levels of reactive oxygen species and nitric oxide may lead to the disruption of endothelial cells and blood-brain barrier breakdown. Overall, in our case, we think chronic alcoholism and alcohol withdrawal might have caused endothelial dysfunction leading to PRES.

Salvianolic acid B improves the disruption of high glucose-mediated brain microvascular endothelial cells via the ROS/HIF-1α/VEGF and miR-200b/VEGF signaling pathways

The study investigated the roles and mechanisms of Salvianolic acid B (Sal B) on permeability of rat brain microvascular endothelial cells (RBMECs) exposed to high glucose. The results demonstrated that Sal B greatly up-regulated the expression of tight junction (TJ) proteins and decreased the permeability of RBMECs compared with the control group. And the increase of reactive oxidative species (ROS) production, the upregulation of hypoxia-inducible factor-1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF) protein induced by high glucose were antagonized by Sal B. In addition, a great decrease of microRNA-200b (miR-200b) was observed in the RBMECs under high-glucose condition, which was significantly increased by Sal B pretreatment. And overexpression of miR-200b markedly attenuated the RBMECs permeability and inhibited the expression of VEGF protein by targeting with 3′-UTR of its mRNA. This led to the conclusion that Sal B-mediated improvement of blood-brain barrier dysfunction induced by high-glucose is related to the ROS/HIF-1α/VEGF and miR-200b/VEGF signaling pathways.

Ketamine alleviates bradykinin-induced disruption of the mouse cerebrovascular endothelial cell-constructed tight junction barrier via a calcium-mediated redistribution of occludin polymerization.

Following brain injury, a sequence of mechanisms leads to disruption of the blood-brain barrier (BBB) and subsequent cerebral edema, which is thought to begin with activation of bradykinin. Our previous studies showed that ketamine, a widely used intravenous anesthetic agent, can suppress bradykinin-induced cell dysfunction. This study further aimed to evaluate the protective effects of ketamine against bradykinin-induced disruption of the mouse cerebrovascular endothelial cell (MCEC)-constructed tight junction barrier and the possible mechanisms. Exposure of MCECs to bradykinin increased intracellular calcium (Ca2+) concentrations in a time-dependent manner. However, pretreatment of MCECs with ketamine time- and concentration-dependently lowered the bradykinin-induced calcium influx. As to the mechanisms, although exposure of MCECs to ketamine induced bradykinin R1 receptor protein and mRNA expression, this anesthetic did not change levels of the bradykinin R2 receptor, a major receptor that responds to bradykinin stimulation. Bradykinin increased amounts of soluble occludin in MCECs, but pretreatment with ketamine alleviated this disturbance in occludin polymerization. Consequently, exposure to bradykinin decreased the transendothelial electronic resistance in the MCEC-constructed tight junction barrier. However, pretreatment with ketamine attenuated the bradykinin-induced disruption of the tight junction barrier. Taken together, this study shows that ketamine at a therapeutic concentration can protect against bradykinin-induced breakage of the BBB via suppressing calcium-dependent redistribution of occludin tight junctions. Thus, ketamine has the potential for maintaining the BBB in critically ill patients with severe brain disorders.

Activation of classical estrogen receptor subtypes reduces tight junction disruption of brain endothelial cells under ischemia/reperfusion injury

Ischemic stroke, which induces oxidative stress in the brain, disrupts tight junctions (TJs) between brain endothelial cells, resulting in blood–brain barrier (BBB) breakdown and brain edema. Estrogen reduces oxidative stress and protects brain endothelial cells from ischemic insult. The aim of this study was to determine the protective effects of estrogen on TJ disruption and to examine the roles of classical estrogen receptor (ER) subtypes, ERα- and ERβ, in estrogen effects in brain endothelial cells (bEnd.3) exposed to oxygen-glucose deprivation/reperfusion (OGD/R) injury. Estrogen pretreatment prevented OGD/R-induced decreases in cell viability and TJ protein levels. ERα- and ERβ-specific agonists also reduced TJ disruption. Knockdown of ERα or ERβ expression partially inhibited the effects of estrogen, but completely reversed the effects of corresponding ER subtype-specific agonists on the outcomes of OGD/R. During the early reperfusion period, activation of extracellular signal-regulated kinase1/2 and hypoxia-inducible factor 1α/vascular endothelial growth factor was associated with decreased expression of occludin and claudin-5, respectively, and these changes in TJ protein levels were differentially regulated by ER subtype-specific agonists. Our results suggest that ERα and ERβ activation reduce TJ disruption via inhibition of signaling molecules after ischemic injury and that targeting each ER subtype can be a useful strategy for protecting the BBB from ischemic stroke in postmenopausal women.

Pseudomonas aeruginosa exoenzymes U and Y induce a transmissible endothelial proteinopathy.

We tested the hypothesis that Pseudomonas aeruginosa type 3 secretion system effectors exoenzymes Y and U (ExoY and ExoU) induce release of a high-molecular-weight endothelial tau, causing transmissible cell injury characteristic of an infectious proteinopathy. Both the bacterial delivery of ExoY and ExoU and the conditional expression of an activity-attenuated ExoU induced time-dependent pulmonary microvascular endothelial cell gap formation that was paralleled by the loss of intracellular tau and the concomitant appearance of high-molecular-weight extracellular tau. Transfer of the high-molecular-weight tau in filtered supernatant to naïve endothelial cells resulted in intracellular accumulation of tau clusters, which was accompanied by cell injury, interendothelial gap formation, decreased endothelial network stability in Matrigel, and increased lung permeability. Tau oligomer monoclonal antibodies captured monomeric tau from filtered supernatant but did not retrieve higher-molecular-weight endothelial tau and did not rescue the injurious effects of tau. Enrichment and transfer of high-molecular-weight tau to naïve cells was sufficient to cause injury. Thus we provide the first evidence for a pathophysiological stimulus that induces release and transmissibility of high-molecular-weight endothelial tau characteristic of an endothelial proteinopathy.

Raf/ERK drives the proliferative and invasive phenotype of BMPR2-silenced pulmonary artery endothelial cells.

A proliferative endothelial cell phenotype, inflammation, and pulmonary vascular remodeling are prominent features of pulmonary arterial hypertension (PAH). Bone morphogenetic protein type II receptor (BMPR2) loss-of-function is the most common cause of heritable PAH and has been closely linked to the formation of pathological plexiform lesions. Although some BMPR2 mutations leave ligand-dependent responses intact, the disruption of ligand-independent, noncanonical functions are universal among PAH-associated BMPR2 genotypes, but incompletely understood. This study examined the noncanonical signaling consequences of BMPR2 silencing in human pulmonary artery endothelial cells to identify potential therapeutic targets. BMPR2 siRNA silencing resulted in a proliferative, promigratory pulmonary artery endothelial cell phenotype and disruption of cytoskeletal architecture. Expression profiling closely reflected these phenotypic changes. Gene set enrichment and promoter analyses, as well as the differential expression of pathway components identified Ras/Raf/ERK signaling as an important consequence of BMPR2 silencing. Raf family members and ERK1/2 were constitutively activated after BMPR2 knockdown. Two Raf inhibitors, sorafenib and AZ628, and low-dose nintedanib, a triple receptor tyrosine kinase inhibitor upstream from Ras, reversed the abnormal proliferation and hypermotility of BMPR2 deficiency. Inhibition of dysregulated Ras/Raf/ERK signaling may be useful in reversing vascular remodeling in PAH.

Relationship of ultrasound cavitation pressure amplitude and rat blood brain barrier permeability

Objective To explore the effectiveness of different peak negative pressure ultrasound irradiated microbubble destruction on the control of rat blood brain barrier permeability. Methods Thirty healthy SD rats were divided into 5 groups according to different ultrasound pressure level (62 kPa, 86 kPa, 181 kPa, 324 kPa, 708 kPa). After intravenous injection of microbubble, the rats were underwent ultrasonic irradiation for 9 minutes, and the contralateral hemispheres were served as controls. The permeability of blood brain barrier was determined by the quantitative analysis of Evans blue and lanthanum nitrate extravasation. Results Quantitative analysis results revealed a leakage in Evans blue about(4.52±0.11) μg/g and (10.56±0.13)μg/g in group 181 kPa and 708 kPa, respectively. A wide endothelial cell gap with lanthanum nitrate trace leaked out was shown at the group of ultrasound pressure of 181 kPa, and erythrocytes extravasations were found when ultrasound pressure at 708 kPa. Conclusions The pressure amplitude in microbubble enhanced cavitation can effectively regulate and control blood-brain barrier opening in rats. Key words: Blood-brain barrier; Microbubblest; Cavitation; Hemicerebrum; Rat

Abl Family Kinases Differentially Regulate LPS‐induced Endothelial Barrier Dysfunction

Rationale: Acute Lung Injury (ALI) involves inflammation-induced disruption of the pulmonary endothelial cell (EC) barrier. Abl family kinases, c-Abl and Arg, have distinct roles in barrier regulation in response to thrombin, histamine, VEGF and oxidative stress. Our prior work indicates that the Abl kinase inhibitor imatinib protects against LPS-induced ALI. To elucidate the underlying mechanisms, we investigated the roles of c-Abl and Arg in LPS-induced EC dysfunction. Methods Intercellular gap formation was quantified in human pulmonary artery ECs treated with imatinib (20 μM) and LPS (1 μg/mL) via FITC-avidin binding to biotinylated gelatin. Western blots and immunofluorescence microscopy were conducted on LPS challenged ECs to assess c-Abl/Arg expression, kinase activity (pCrkL), and sub-cellular localization. Additionally, siRNA was used to assess the roles of c-Abl/Arg in EC contractile apparatus localization and LPS-induced EC activation (VCAM-1, IL-8). Results C-Abl/Arg inhibition attenuated LPS-induced gap formation (22%, p=0.01). LPS increased c-Abl/Arg activation, without altering expression. However, only c-Abl co-localized with actin stress fibers after LPS. Additionally, only c-Abl silencing attenuated LPS-induced VCAM-1 expression (33%, p=0.04) and IL-8 release (48%, p=0.048). Furthermore, the actinomyosin contractile apparatus was localized centrally in c-Abl-silenced ECs, but peripherally in Arg-silenced ECs. Conclusions Abl family kinases differentially regulate EC dysfunction in response to LPS. The FDA approved inhibitors of these kinases may represent potential therapeutic options for ALI.

Neuraminidase Enhances Pseudomonas Aeruginosa Secretome Disruption of the Rat Pulmonary Microvascular Endothelial Cell Monolayer

Neuraminidase (NA), an enzyme that removes sialic acids and is produced by many pathogenic organisms, has previously been shown to cause endothelial barrier disruption. P. aeruginosa strains secrete a variety of proteins, including NA, into the extracellular environment, the collection of which may be termed the secretome. Many of these proteins are virulence factors and expression can vary across strains, as well as over the course of growth and in response to environmental factors. For example, strain PAO1 expresses LasB, an elastase which has been previously implicated in disruption of endothelial monolayers by cleaving VE‐cadherin, which is sialylated, but strain Pa103 does not. This study investigates the role of NA in P. aeruginosa secretome disruption of the pulmonary microvascular endothelial cell (PMVEC) monolayer. PMVEC monolayers were treated with secretome from various P. aeruginosa strains including the wildtype PAO1, a NA deleted strain, PAO1Δ2794, and the wildtype strain Pa103. Secretomes from the different strains caused gap formation in the PMVEC monolayer at differing times. Large gaps were observed at 18 hours after treatment with PAO1 secretome, while similar damage from the PAO1Δ2794 secretomes was not observed until 26 hours after treatment. Only minor damage was observed upon treatment with Pa103 secretome at any time point. The delay in damage observed between the wild‐type secretome treatment and the NA‐deleted secretome lead to the conclusion that NA enhances disruption of the PMVEC barrier by P. aeruginosa PAO1 secretome. Supported by R01HL107778.

Pseudomonas aeruginosa Exoenzymes U and Y Induce a Transmissible Endothelial Tauopathy

Here, we tested the hypothesis that Pseudomonas aeruginosa type 3 secretion system effectors ExoY and ExoU induce endothelial tau oligomerization and release, causing transmissible cytotoxicity characteristic of an infectious tauopathy. Both the bacterial delivery of ExoY and ExoU, and the conditional expression of an activity‐attenuated ExoU, induced time‐dependent pulmonary microvascular endothelial cell (PMVEC) gap formation that was paralleled by loss of intracellular tau and concomitant appearance of extracellular tau oligomers. Transfer of tau oligomers in filtered supernatant onto naïve endothelial cells resulted in the intracellular accumulation of oligomeric tau inclusions accompanied by cytotoxicity, as determined by inter‐endothelial gap formation and decreased endothelial network stability in Matrigel. Tau oligomer monoclonal antibodies captured monomeric tau from filtered supernatant, but did not retrieve higher molecular mass tau oligomers, and did not rescue the cytotoxic effects of tau. Since cytotoxic effects were not rescued, we combined an electrophoresis and electroelution approach to recover the high molecular mass tau oligomers, treat PMVECs with these oligomers, and recapitulate cytotoxicity. Thus, we provide the first evidence for a pathophysiological stimulus that induces endothelial tau oligomerization, release and transmissible cytotoxicity, characteristic of an endothelial tauopathy.

P026 Mechanical ventilation promotes lung metastasis in 4T1 breast cancer lung-metastasized models

Goals: Recent evidence suggests that general anaesthesia and perioperative factors significantly increased risk of cancer recurrence compared to locoregional anaesthesia in patients undergoing surgery, however, their effects on behavior of cancer cells are not yet known. The endothelial cell and epithelial disruption was observed during mechanical ventilation, general anaesthesia in animalmodels. This studywas to test the hypothesis thatmechanical ventilation during cancer surgery induces lung stroma/tissue milieu changes, creating a favorable microenvironment for postoperative lung metastatic tumor establishment. Methods: Female BALB/c mice were anesthetized by intraperitoneal pentobarbital administration and divided into a mechanical ventilation group [mice ventilated with low pressures and low tidal volumes, 60min; n =8) and a control group (no ventilation; n =8). All the mice were subjected to intravenous injection of murine mammary adenocarcinoma 4T1-green fluorescent protein (GFP) 10 cells during anaesthesia. 24 hr later, sections from the lung tissue were examined and 4T1 celler were counted by direct GFP fluorescence observation, hematoxylineosin histology and Ep-CAM immunohistochemistry. To closely mimic human cancer surgical and anaesthetic manipulation, A clinically relevant animal model of spontaneous breast cancer lung metastasis with surgical resection of primary tumor was used to investigate the purified mechanical ventilation event that dictate lung metastasis post-operation. Female BALB/c mice were inoculated with metastatic murine mammary adenocarcinoma 4T1 cells in the mammary fat pad (3×105/mouse). After 14 day growth, mice were anesthetized by intraperitoneal pentobarbital administration and divided into a mechanical ventilation group [mice ventilated with low pressures and low tidal volumes, 60min; n =6) and a control group (no ventilation; n =6). All the mice were subjected to flank tumor resection during anaesthesia. Two weeks later, Metastatic tumor burden was assessed by both macroscopic metastatic nodule count and hematoxylin-eosin histology. Results: Mechanical ventilation was associated with increased circulating breast tumor cells arrest at microvasculature of lung (P = 0.01). The histology analysis data indicated that mechanical ventilation induced endothelial-epithelium abnormalities, inflammatory reaction and significantly upregulated the expression of epithelial adhesion molecules (Ep-CAM). Postoperative metastases exhibited significantly increase in mechanical ventilated groups in comparison with those in non-ventilated group (P < 0.05). Conclusion: Mechanical ventilation led to a progressive phenotype of lung metastases that exhibited a microenvironment for cancer cell adhesion, invasion, survive and growth within lungs. Disclosure of Interest: No significant relationships. P027 The analysis of cytogenetic intra-tumoral heterogeneity in squamous cell carcinoma of the breast M. Oikawa *, A. Igawa, M. Ishida, Y. Nakamura, S. Nishimura, C. Koga, S. Akiyoshi, Y. Koi, K. Taguchi, S. Ohno. Breast Oncology, National Kyushu Cancer Center, Fukuoka, Japan, Pathology, National Kyushu Cancer Center, Fukuoka, Japan

Red light has the capacity to blunt corneal endothelial cell damage in situ.

AbstractPurpose To cause damage to rat corneal endothelial cells in situ and determine whether exposure to red light blunts this influence.Methods Corneal endothelial cells from one eye in anesthetised rats were damaged by placing a needle in the anterior chamber and elevating the intraocular pressure (IOP, approximately 130mm Hg) for 60 minutes in the dark. In some instances the cornea was exposed to red light (2500 lux). Rats were either killed immediately or after periods of up to 15 days. Flat mounts and transverse sections of cornea were processed for the localisation of tight junctional protein ZO‐1 and JC‐1 to monitor mitochondrial health with their degree of depolarization indicated by changes in the red to green intensity of fluorescence.Results Raise IOP causes damage to the mosaic appearance of corneal endothelial cells clearly indicated by staining for ZO‐1. Exposure to red light during elevation of IOP reduced the amount of patchy damage to endothelial cells. Corneal endothelial cells appear green when stained with JC‐1 but after 60 minutes of raised IOP the cytoplasm of many cells appear red. Both the numbers and intensity of red endothelial cells increased in JC‐1 stained corneas exposed to red light. Three days after an insult of raised IOP, some red JC‐1 staining was present only in corneas exposed to red light.Conclusion Raised IOP causes damage and disruption of corneal endothelial cells. This process is blunted by red light. JC‐1 staining suggests that endothelial cell mitochondria are activated by raised IOP (presumably as a protective mechanism) and that this process is enhanced by red light.

PKC-dependent phosphorylation of eNOS at T495 regulates eNOS coupling and endothelial barrier function in response to G+ -toxins.

Gram positive (G+) infections make up ∼50% of all acute lung injury cases which are characterized by extensive permeability edema secondary to disruption of endothelial cell (EC) barrier integrity. A primary cause of increased permeability are cholesterol-dependent cytolysins (CDCs) of G+-bacteria, such as pneumolysin (PLY) and listeriolysin-O (LLO) which create plasma membrane pores, promoting Ca2+-influx and activation of PKCα. In human lung microvascular endothelial cells (HLMVEC), pretreatment with the nitric oxide synthase (NOS) inhibitor, ETU reduced the ability of LLO to increase microvascular cell permeability suggesting an endothelial nitric oxide synthase (eNOS)-dependent mechanism. LLO stimulated superoxide production from HLMVEC and this was prevented by silencing PKCα or NOS inhibition suggesting a link between these pathways. Both LLO and PLY stimulated eNOS T495 phosphorylation in a PKC-dependent manner. Expression of a phosphomimetic T495D eNOS (human isoform) resulted in increased superoxide and diminished nitric oxide (NO) production. Transduction of HLMVEC with an active form of PKCα resulted in the robust phosphorylation of T495 and increased peroxynitrite production, indicative of eNOS uncoupling. To determine the mechanisms underlying eNOS uncoupling, HLMVEC were stimulated with LLO and the amount of hsp90 and caveolin-1 bound to eNOS determined. LLO stimulated the dissociation of hsp90, and in particular, caveolin-1 from eNOS. Both hsp90 and caveolin-1 have been shown to influence eNOS uncoupling and a peptide mimicking the scaffolding domain of caveolin-1 blocked the ability of PKCα to stimulate eNOS-derived superoxide. Collectively, these results suggest that the G+ pore-forming toxins promote increased EC permeability via activation of PKCα, phosphorylation of eNOS-T495, loss of hsp90 and caveolin-1 binding which collectively promote eNOS uncoupling and the production of barrier disruptive superoxide.

C0228: Platelet Thromboxane A2 is an Extracellular Mediator of the Prothrombotic Effect of Erythrocytes

Background: The thrombotic cellular mechanisms associated with cardiovascular events remains unclear, largely because of an inability to visualize thrombus formation. In addition, the contribution of endothelial cell (EC) injuries to thrombus formation processes are unclear. Methods: We developed in vivo fluorescent imaging technique based on singleand multi-photon microscopy to revealed the multicellular processes during thrombus development. We utilized multi-color and high-speed resonance scanner systems, and applied to fluorescent protein transgenic mice. We could identify single platelet kinetics in developing thrombus in living animals. Results: We assessed dynamic cellular interplay in two thrombosis models. First, we visualized that rapidly developing thrombi composed of discoid platelets without EC disruption was triggered by reactive oxygen species by photochemically induction from moderate power laser irradiation. In this model, thrombus consisted by discoid platelet aggregations without leukocyte recruitment. The other model is, thrombus with EC disruption. High power laser induced EC erosion and extravasations of circulating leukocytes with thrombus development. Inflammatory cytokine, adhesion molecules dynamically control these two processes. As for the thrombus formation with EC disruption, chemokine expressions in endothelium and leukocyte (especially neutrophils) recruitment played a significant role in these processes. TLR4 signalling also contributed to these steps. Conclusions: In sum, using our imaging system can be a powerful tool to analyze thrombus formation and evaluate the therapeutic strategies.

Curcumin-primed exosomes mitigate endothelial cell dysfunction during hyperhomocysteinemia

AimExosomes, the nano-units (<200nm), released from diverse cell types in the extracellular body fluid, possess non-immunogenic property and ability to cross the blood–brain barrier (BBB). Since exosomes carry biological information from their cells of origin, we hypothesize that priming cells with potential therapeutic agents release improved cellular contents through exosomes. Curcumin possesses anti-oxidative and anti-inflammatory properties and provides a promising treatment for cerebral diseases and therefore, the aim of the study is to establish that mouse brain endothelial cells (MBECs) when primed with curcumin (7.5μM), release an alleviated exosome population that can help recover the endothelial cell (EC) layer permeability. Main methodsHomocysteine is a well-known causative factor of BBB disruption; therefore, homocysteine-treated ECs were used as a model of BBB disruption and curcumin-primed exosomes were utilized to check their potential for mitigating EC disruption. MBECs were treated with curcumin and exosomes were isolated by using ultracentrifugation and immunoprecipitation. Expression levels of junction proteins were detected by Western blot and immunocytochemistry assays. Endothelial cell permeability was analyzed with Fluorescein isothiocyanate-Bovine serum albumin (FITC-BSA) leakage assay using transwell permeable supports. Key findingsExosomes derived from curcumin-treated (primed) cells (CUR-EXO) alleviated oxidative stress, tight junctions (ZO-1, claudin-5, occludin), adherent junction (VE-cadherin) proteins and EC layer permeability induced during EC damage due to high homocysteine levels (hyperhomocysteinemia). SignificanceIn conclusion, the study potentiates the use of CUR-EXO for cerebral diseases where drug delivery is still a challenge. The results also pave the way to novel translational therapies for cerebral diseases by maintaining and establishing therapeutic conservatories via primed exosomes.

The effects of low-frequency ultrasound and microbubbles on rabbit hepatic tumors

High-intensity focused ultrasound in combination with microbubbles (MBs) is able to inhibit the growth of VX2 rabbit liver tumors in vivo and prolong the survival time of the animals. In this study, we attempt to investigate the feasibility of VX2 tumor growth inhibition using low-frequency ultrasound (US)-mediated MB disruption. Forty-eight New Zealand rabbits with hepatic VX2 tumors were divided into four groups: control, MBs group, low-frequency US group, and US + MB group. The parameters of the US were 20 kHz, 2 W/cm², 40% duty cycle, 5 min, and once every other day for 2 weeks. At the end of the therapy experiment, 24 rabbits were euthanized, and the cancers were collected and cut into five sections for histological examination, immunohistochemistry, laser confocal microscopy, western blotting assays, and transmission electron microscopy (TEM). Another 24 rabbits were saved, and overall survival time was recorded. The tumor volumes in control, MB, US, and US + MB groups were 6.36 ± 0.58, 5.68 ± 0.42, 5.29 ± 0.26, and 2.04 ± 0.14 cm³, respectively (US + MB versus the other three groups, P < 0.01). Tumor cells manifested coagulation necrosis with internal calcification. Hematoxylin and eosin (H–E) staining revealed interstitial hemorrhage and intravascular thrombosis. The intensity of cyclooxygenase-2 (COX-2), and vascular endothelial growth factor (VEGF) in the US + MB group in the immunohistochemical staining, laser confocal microscopy, and western blotting assays was lower than that of the other three groups (P < 0.05). TEM of the US + MB group revealed vascular endothelial cell wall rupture, widened endothelial cell gaps, interstitial erythrocyte leakage, and microvascular thrombosis, while intact vascular endothelial cells and normal erythrocytes in the tumor vessels were observed in control, MB, and US groups. Rabbits treated with US + MB had a significantly longer overall survival than those in the other three groups (χ2 = 9.328, P = 0.0242). VX2 tumor growth could be inhibited by cavitation induced using low-frequency US and MB.

In vitro inhibition of protease-activated receptors 1, 2 and 4 demonstrates that these receptors are not involved in an Acanthamoeba castellanii keratitis isolate-mediated disruption of the human brain microvascular endothelial cells

Granulomatous amoebic encephalitis is a rare but serious human disease leading almost always to death. The pathophysiology of amoebic encephalitis is better understood, while events leading to the constitution of brain infection are largely unknown. Traversal of the blood–brain barrier is a key step in amoebae invasion of the central nervous system and facilitated by amoebic extracellular proteases. By using specific inhibitors of protease-activated receptors 1, 2 and 4, here we studied the role of these host receptors in Acanthamoeba castellanii-mediated damage to human brain microvasculature endothelial cells (HBMEC), which constitute the blood–brain barrier. The primary HBMEC were incubated with A. castellanii-conditioned medium in the presence or absence of FR-171113 (selective inhibitor of protease-activated receptor 1), FSLLRY-NH2 (inhibitor of protease-activated receptor 2), and tcY-NH2 (inhibitor of protease-activated receptor 4). The HBMEC monolayer disruptions were assessed by microscopy using Eosin staining, while host cell cytotoxicity was determined by measuring the release of cytoplasmic lactate dehydrogenase. Zymographic assays were performed to determine the effects of inhibitors of protease-activated receptors on the extracellular proteolytic activities of A. castellanii. A. castellanii-conditioned medium produced severe HBMEC monolayer disruptions within 60min. The selective inhibitors of protease-activated receptors tested did not affect HBMEC monolayer disruptions. On the contrary, pre-treatment of A. castellanii-conditioned medium with phenylmethylsulfonyl fluoride, a serine protease inhibitor, or heating for 10min at 95°C abolished HBMEC monolayer disruptions. Additionally, inhibitors of protease-activated receptors tested, failed to block A. castellanii-mediated HBMEC cytotoxicity and did not affect extracellular proteolytic activities of A. castellanii. Protease-activated receptors 1, 2 and 4 do not appear to play a role in A. castellanii-mediated dysfunction of HBMEC, which constitute the blood–brain barrier. The role of additional protease-activated receptors in amoebic invasion of the central nervous system is discussed further.

Thrombus Development Processes Are Dependent On Endothelial Injuries: Examined By In Vivo Molecular Imaging

The thrombotic cellular mechanisms associated with cardiovascular events remains unclear, largely because of an inability to visualize thrombus formation. In addition, the contribution of endothelial cell (EC) injuries to thrombus formation processes are unclear, and we developed in vivo imaging technique based on single- and multi-photon microscopy to revealed the multicellular processes during thrombus development (Figure a,b). We visualized the cell dynamics including single platelet behavior, and assessed dynamic cellular interplay in two thrombosis models. First, we visualized that rapidly developing thrombi composed of discoid platelets without EC disruption was triggered by ROS photochemically induced by moderate power laser irradiation (Figure c). In this model, thrombus consisted by discoid platelet aggregations without leukocyte recruitment. The second model is, thrombus with EC disruption. High power laser induced EC erosion and extravasations of circulating leukocytes with thrombus development. Inflammatory cytokine, adhesion molecules dynamically control these two processes. (Figure d) [Display omitted] Using this technique, we elucidated that Lnk (adapter protein) regulates integrin signaling leading to stabilization of developing thrombus without EC disruption. Specifically, adhesion molecules dynamically control these processes. Thrombus formation was initiated by the binding of platelet GPIb-alpha to endothelial von Willebrand Factor in this model, and actin linker talin-dependent activation of alphaIIb-beta3 integrin in platelets was required for late phase thrombus stability.As for the thrombus formation with EC disruption, chemokine expressions in endothelium and leukocyte (especially neutrophils) recruitment played a significant role in these processes. TLR4 signaling also contributed to these steps.In sum, using our imaging system can be a powerful tool to analyze thrombus formation and evaluate the therapeutic strategies. Disclosures:No relevant conflicts of interest to declare.