Nanomedicine: Patuletin-conjugated with zinc oxide exhibit potent effects against Gram-negative and Gram-positive bacterial pathogens.

With the emergence of drug-resistance, there is a need for novel anti-bacterials or to enhance the efficacy of existing drugs. In this study, Patuletin (PA), a flavanoid was loaded onto Gallic acid modified Zinc oxide nanoparticles (PA-GA-ZnO), and evaluated for antibacterial properties against Gram-positive (Bacillus cereus and Streptococcus pneumoniae) and Gram-negative (Samonella enterica and Escherichia coli) bacteria. Characterization of PA, GA-ZnO and PA-GA-ZnO' nanoparticles was accomplished utilizing fourier-transform infrared spectroscopy, efficiency of drug entrapment, polydispersity index, zeta potential, size, and surface morphology analysis through atomic force microscopy. Using bactericidal assays, the results revealed that ZnO conjugation displayed remarkable effects and enhanced Patuletin's effects against both Gram-positive and Gram-negative bacteria, with the minimum inhibitory concentration observed at micromolar concentrations. Cytopathogenicity assays exhibited that the drug-nanoconjugates reduced bacterial-mediated human cell death with minimal side effects to human cells. When tested alone, drug-nanoconjugates tested in this study showed limited toxic effects against human cells in vitro. These are promising findings, but future work is needed to understand the molecular mechanisms of effects of drug-nanoconjugates against bacterial pathogens, in addition to in vivo testing to determine their translational value. This study suggests that Patuletin-loaded nano-formulation (PA-GA-ZnO) may be implicated in a multi-target mechanism that affects both Gram-positive and Gram-negative pathogen cell structures, however this needs to be ascertained in future work.

The synthetic cannabinoid 5-fluoro ABICA upregulates angiogenic markers and stimulates tube formation in human brain microvascular endothelial cells.

Objective: Synthetic cannabinoids (SCs), a class of psychoactive compounds emulating the effects of natural cannabis, have prompted addiction and psychosis concerns. However, recent research has suggested potential pharmacological applications, particularly in brain angiogenesis-an essential physiological process for growth, repair, and tissue maintenance, in which new blood vasculature is formed from existing vasculature. This study explored the in vitro ability of the SC 5-fluoro ABICA to enhance new blood formation processes in human brain microvascular endothelial cells (HBMECs). Methods: HBMECs were treated with various concentrations of 5-fluoro ABICA (1 µM, 0.1 µM, 0.01 µM, 0.001 µM, and 0.0001 µM). A comprehensive analysis was conducted, including MTT assays indicating cell viability, wound healing assays indicating migration ability, and tube formation assays indicating the angiogenesis potential of endothelial cells. Additionally, mRNA expression and protein levels of specific pro-angiogenic factors were measured, and the phosphorylation levels of glycogen synthase kinase-3ß were detected in treated HBMECs through ELISA, real-time PCR, and western blotting. Results: Treatment with 5-fluoro ABICA effectively stimulated proliferation, migration, and tube formation in HBMECs in a dose-dependent manner; markedly increased the expression of pro-angiogenic factors; and upregulated levels of phosphorylated-GSK-3ß. Conclusion: Our findings demonstrate that 5-fluoro ABICA stimulates angiogenesis in endothelial cells, thus potentially offering therapeutic options for diseases associated with angiogenesis. However, further research is needed to fully understand the molecular mechanism of 5-fluoro ABICA in angiogenesis, including ethical considerations regarding its use in medical research.

Breast adenocarcinoma cells adhere stronger to brain pericytes than to endothelial cells.

Most of the malignancies detected within the brain parenchyma are of metastatic origin. As the brain lacks classical lymphatic circulation, the primary way for metastasis relies on hematogenous routes. Dissemination of metastatic cells to the brain implies attachment to the luminal surface of brain endothelial cells, transmigration through the vessel wall, and adhesion to the brain surface of the vasculature. During this process, tumor cells must interact with brain endothelial cells and later on with pericytes. Physical interaction between tumor cells and brain vascular cells might be crucial in the successful extravasation of metastatic cells through blood vessels and later in their survival within the brain environment. Therefore, we applied single-cell force spectroscopy to investigate the nanoscale adhesive properties of living breast adenocarcinoma cells to brain endothelial cells and pericytes. We found target cell type-dependent adhesion characteristics, i.e. increased adhesion of the tumor cells to pericytes in comparison to endothelial cells, which underlines the existence of metastatic potential-related nanomechanical differences relying partly on membrane tether dynamics. Varying adhesion strength of the tumor cells to different cell types of brain vessels presumably reflects the transitory adhesion to endothelial cells before extravasation and the long-lasting strong interaction with pericytes during survival and proliferation in the brain. Our results highlight the importance of specific mechanical interactions between tumor cells and host cells during metastasis formation.

Perillyl alcohol modulates activation, permeability and integrity of human brain endothelial cells induced by Plasmodium falciparum

BACKGROUND Cerebral malaria (CM) is a severe immunovasculopathy caused for Plasmodium falciparum infection, which is characterised by the sequestration of parasitised red blood cells (pRBCs) in brain microvessels. Previous studies have shown that some terpenes, such as perillyl alcohol (POH), exhibit a marked efficacy in preventing cerebrovascular inflammation, breakdown of the brain-blood barrier (BBB) and brain leucocyte accumulation in experimental CM models. OBJECTIVE To analyse the effects of POH on the endothelium using human brain endothelial cell (HBEC) monolayers co-cultured with pRBCs. METHODOLOGY The loss of tight junction proteins (TJPs) and features of endothelial activation, such as ICAM-1 and VCAM-1 expression were evaluated by quantitative immunofluorescence. Microvesicle (MV) release by HBEC upon stimulation by P. falciparum was evaluated by flow cytometry. Finally, the capacity of POH to revert P. falciparum-induced HBEC monolayer permeability was examined by monitoring trans-endothelial electrical resistance (TEER). FINDINGS POH significantly prevented pRBCs-induced endothelial adhesion molecule (ICAM-1, VCAM-1) upregulation and MV release by HBEC, improved their trans-endothelial resistance, and restored their distribution of TJPs such as VE-cadherin, Occludin, and JAM-A. CONCLUSIONS POH is a potent monoterpene that is efficient in preventing P. falciparum-pRBCs-induced changes in HBEC, namely their activation, increased permeability and alterations of integrity, all parameters of relevance to CM pathogenesis.

Simplified in vitro 3D co-culture-based blood-brain barrier model using transwell.

The blood-brain barrier (BBB) is a major hurdle for treatment of brain diseases. To overcome this, precise and reproducible BBB model is one of the key factors for successful evaluation of BBB-penetrating efficacy of developmental drugs. Thus, in vitro BBB model recapitulating the physiological structure of the BBB is a valuable tool for drug discovery and development for brain diseases. Here, we develop a simplified 3D co-culture-based BBB model using immortalized human brain endothelial cells and immortalized human astrocytes mixed with Matrigel allowing model preparation within 30 min. We directly compare our 3D BBB model to a 2D BBB model comprised solely of immortalized brain endothelial cells, to demonstrate that our 3D BBB model blocks penetration of Dextran molecules with various molecular weights, remain durable and impermeable even in a BBB-degrading condition, and rapidly form tight junctions while the 2D BBB model do not. In conclusion, this establishes our simplified 3D BBB model as a valuable tool for high throughput screening of drug candidates for brain diseases.

Mechanisms underlying protective effects of vitamin E against mycotoxin deoxynivalenol-induced oxidative stress and its related cytotoxicity in primary human brain endothelial cells.

Fusarium mycotoxins are one of the largest families of mycotoxins. Among these mycotoxins, deoxynivalenol is the most widespread pollutant of grains. However, the mechanism underlying the effect of deoxynivalenol on cytotoxicity in human brain endothelial cells was still unclear. This study examined whether deoxynivalenol induced oxidative stress-associated cytotoxicity in primary human brain endothelial cells (HBEC-5i), and explored whether Vitamin E (VE), a selective antioxidant, had protective effects on deoxynivalenol-treated cells. Deoxynivalenol (10-50 µM) concentration-dependently induced cytotoxicity in HBEC-5i cells. Deoxynivalenol (IC50 = 20 µM) activated mitochondrial apoptotic pathway by modulating antioxidant protein expressions (Nrf2, HO-1 and NQO1). More significantly, pre-treatment with VE (20 µM) attenuated the deoxynivalenol-induced cytotoxicity in this cell model. Together, VE significantly alleviated the apoptotic effects of deoxynivalenol in HBEC-5i cells suggesting that it protected the cells against deoxynivalenol-induced oxidative damage. Our findings provided new insight that VE had the potential to ameliorate neurotoxicity of deoxynivalenol.

CdSe/ZnS Core-Shell-Type Quantum Dot Nanoparticles Disrupt the Cellular Homeostasis in Cellular Blood-Brain Barrier Models.

Two immortalized brain microvascular endothelial cell lines (hCMEC/D3 and RBE4, of human and rat origin, respectively) were applied as an in vitro model of cellular elements of the blood-brain barrier in a nanotoxicological study. We evaluated the impact of CdSe/ZnS core-shell-type quantum dot nanoparticles on cellular homeostasis, using gold nanoparticles as a largely bioorthogonal control. While the investigated nanoparticles had surprisingly negligible acute cytotoxicity in the evaluated models, a multi-faceted study of barrier-related phenotypes and cell condition revealed a complex pattern of homeostasis disruption. Interestingly, some features of the paracellular barrier phenotype (transendothelial electrical resistance, tight junction protein gene expression) were improved by exposure to nanoparticles in a potential hormetic mechanism. However, mitochondrial potential and antioxidant defences largely collapsed under these conditions, paralleled by a strong pro-apoptotic shift in a significant proportion of cells (evidenced by apoptotic protein gene expression, chromosomal DNA fragmentation, and membrane phosphatidylserine exposure). Taken together, our results suggest a reactive oxygen species-mediated cellular mechanism of blood-brain barrier damage by quantum dots, which may be toxicologically significant in the face of increasing human exposure to this type of nanoparticles, both intended (in medical applications) and more often unintended (from consumer goods-derived environmental pollution).

Long non­coding RNA SNHG16 inhibits the oxygen­glucose deprivation and reoxygenation­induced apoptosis in human brain microvascular endothelial cells by regulating miR­15a­5p/bcl­2.

MicroRNA (miR) 15a­5p can promote ischemia/reperfusion (I/R)­induced apoptosis of cerebral vascular endothelial cells, which is inhibited by long non­coding RNAs (lncRNAs). The present study investigated the potential of lncRNAs targeting miR­15a­5p to regulate oxygen­glucose deprivation and reoxygenation (OGD­R)­induced apoptosis of human brain microvascular endothelial cells (hBMECs). hBMECs were transfected with or without miR­15a­5p or its mutant, together with p­small nucleolar RNA host gene 16 (SNHG16) or its mutant. Following OGD­R, proliferation, apoptosis and miR­15a­5p, SNHG16 and Bcl­2 expression levels were determined using MTT, flow cytometry, reverse transcription­quantitative PCR or western blotting. The potential interaction of SNHG16 with miR­15a­5p was analyzed by pull­down, luciferase and immunoprecipitation assays. OGD­R induced apoptosis of hBMECs and increased miR­15a­5p expression levels in a time­dependent manner. miR­15a­5p overexpression decreased the proliferation of hBMECs and promoted apoptosis by decreasing Bcl­2 expression levels. SNHG16 was pulled­down by miR­15a­5p and anti­Ago2. miR­15a­5p overexpression significantly decreased SNHG16­regulated luciferase activity and hBMEC survival by increasing apoptosis. SNHG16 overexpression decreased miR­15a­5p expression levels in hBMECs. SNHG16 gradually decreased following OGD­R and its overexpression decreased miR­15a­5p expression levels and promoted the proliferation of hBMECs by decreasing apoptosis. SNHG16 enhanced Bcl­2 expression levels in hBMECs, which was abrogated by miR­15a­5p. Bioinformatics suggest that SNHG16 may antagonize the binding of miR­15a­5p to the 3'UTR of Bcl­2 mRNA. These findings suggest that SNHG16 may protect hBMECs from OGD­R­induced apoptosis by antagonizing the miR­15a­5p/bcl­2 axis. Thus, targeting SNHG16­based mechanisms may provide novel therapeutic strategies for treatment of ischemic stroke.

Zika Virus Persistently Infects and Is Basolaterally Released from Primary Human Brain Microvascular Endothelial Cells.

Zika virus (ZIKV) is a mosquito-borne Flavivirus that has emerged as the cause of encephalitis and fetal microencephaly in the Americas. ZIKV uniquely persists in human bodily fluids for up to 6 months, is sexually transmitted, and traverses the placenta and the blood-brain barrier (BBB) to damage neurons. Cells that support persistent ZIKV replication and mechanisms by which ZIKV establishes persistence remain enigmatic but central to ZIKV entry into protected neuronal compartments. The endothelial cell (EC) lining of capillaries normally constrains transplacental transmission and forms the BBB, which selectively restricts access of blood constituents to neurons. We found that ZIKV (strain PRVABC59) persistently infects and continuously replicates in primary human brain microvascular ECs (hBMECs), without cytopathology, for >9 days and following hBMEC passage. ZIKV did not permeabilize hBMECs but was released basolaterally from polarized hBMECs, suggesting a direct mechanism for ZIKV to cross the BBB. ZIKV-infected hBMECs were rapidly resistant to alpha interferon (IFN-α) and transiently induced, but failed to secrete, IFN-ß and IFN-λ. Global transcriptome analysis determined that ZIKV constitutively induced IFN regulatory factor 7 (IRF7), IRF9, and IFN-stimulated genes (ISGs) 1 to 9 days postinfection, despite persistently replicating in hBMECs. ZIKV constitutively induced ISG15, HERC5, and USP18, which are linked to hepatitis C virus (HCV) persistence and IFN regulation, chemokine CCL5, which is associated with immunopathogenesis, as well as cell survival factors. Our results reveal that hBMECs act as a reservoir of persistent ZIKV replication, suggest routes for ZIKV to cross hBMECs into neuronal compartments, and define novel mechanisms of ZIKV persistence that can be targeted to restrict ZIKV spread.IMPORTANCE ZIKV persists in patients, crossing placental and neuronal barriers, damaging neurons, and causing fetal microencephaly. We found that ZIKV persistently infects brain endothelial cells that normally protect neurons from viral exposure. hBMECs are not damaged by ZIKV infection and, analogous to persistent HCV infection, ZIKV constitutively induces and evades antiviral ISG and IFN responses to continuously replicate in hBMECs. As a result, hBMECs provide a protective niche for systemic ZIKV spread and a viral reservoir localized in the normally protective blood-brain barrier. Consistent with the spread of ZIKV into neuronal compartments, ZIKV was released basolaterally from hBMECs. Our findings define hBMEC responses that contribute to persistent ZIKV infection and potential targets for clearing ZIKV infections from hBMECs. These results further suggest roles for additional ZIKV-infected ECs to facilitate viral spread and persistence in the protected placental, retinal, and testicular compartments.

Laquinimod enhances central nervous system barrier functions.

Laquinimod is currently being tested as a therapeutic drug in multiple sclerosis. However, its exact mechanism of action is still under investigation. Tracking of fluorescently-tagged encephalitogenic T cells during experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, revealed that laquinimod significantly reduces the invasion of pathogenic effector T cells into the CNS tissue. T-cell activation, differentiation and amplification within secondary lymphoid organs after immunization with myelin antigen, their migratory capacity and re-activation within the nervous tissue were either only mildly affected or remained unchanged. Instead, laquinimod directly impacted the functionality of the CNS vasculature. The expression of tight junction proteins p120 and ZO-1 in human brain endothelial cells was up-regulated upon laquinimod treatment, resulting in a significant increase in the transendothelial electrical resistance of confluent monolayers of brain endothelial cells. Similarly, expression of the adhesion molecule activated leukocyte cell adhesion molecule (ALCAM) and inflammatory chemokines CCL2 and IP-10 was suppressed, leading to a significant reduction in the migration of memory TH1 and TH17 lymphocytes across the blood brain barrier (BBB). Our data indicate that laquinimod exerts its therapeutic effects by tightening the BBB and limiting parenchymal invasion of effector T cells, thereby reducing CNS damage.

Effect of N-arachidonoyl-l-serine on human cerebromicrovascular endothelium.

N-arachidonoyl-l-serine (ARA-S) is an endogenous lipid, chemically related to the endocannabinoid, N-arachidonoyl ethanolamine (i.e., anandamide) and with similar physiologic and pathophysiologic functions. Reports indicate that ARA-S possesses vasoactive and neuroprotective properties resembling those of cannabinoids. However, in contrast to cannabinoids, ARA-S binds weakly to its known classical receptors, CB1 and CB2, and is therefore considered to be a 'cannabinoid-like' substance. The originally described ARA-S induced-endothelial-dependent vasorelaxation was not abrogated by CB1, CB2 receptor antagonists or TRPV1 competitive inhibitor. The present report demonstrates that ARA-S enhances the fluorescence staining of both cannabinoid receptors (CB1 and CB2) in human brain endothelial cells (HBEC). This reaction is specific since it was reduced by respective selective receptor antagonist (SR141716A and SR141728A). ARA-S alone or in the presence of ET-1 was shown to alter the cytoskeleton (actin). Both ARA-S stimulated phosphorylation of various kinases (MAPK, Akt, JNK and c-JUN) and alteration of cytoskeleton are mediated via CB1, CB2 and TRPV1 receptors. The findings also showed the involvement of Rho/Rock and PI3/Akt/NO pathways in the ARA-S-induced phosphorylation of kinases and actin reorganization in HBEC. All of the above mentioned ARA-S-induced effects were reduced by the treatment with LY294002 (inhibitor of PI3/Akt kinase), except MAPK kinase. In addition, MAPK, JNK, c-JUN phosphorylation were inhibited by H1152 (inhibitor of Rho/ROCK kinase), except Akt kinase. Furthermore, PI3/Akt pathway was inhibited by pretreatment with l-NAME (inhibitor of NOS). The findings suggest that ARA-S is a modulator of Rho kinase and may play a critical role in the regulation of its activity and subsequent effects on the cytoskeleton and its role in supporting essential cell functions like vasodilation, proliferation and movement.

Marinobufagenin regulates permeability and gene expression of brain endothelial cells.

Marinobufagenin (MBG) is a cardiotonic steroid that increases in the circulation in preeclampsia. Preeclampsia and eclampsia are associated with cerebral edema. Therefore, we examined the effects of MBG on human brain microvascular endothelial cells (HBMEC) in vitro. MBG enhanced the permeability of HBMEC monolayers at 1-, 10-, and 100-nM doses, but had no effect at 0.1 nM. Agilent Human Gene Expression microarrays were utilized in these studies. MBG treatment (10 nM for 12 h) downregulated concentrations of the soluble VEGFR transcript sFLT by 59% but did not alter those of FLTv3 mRNA (determined by quantitative PCR). When treated and control HBMEC transcriptomes were interrogated on microarrays, 1,069 genes appeared to be regulated by MBG. Quantitative RT-PCR confirmed that MBG treatment upregulated ENKUR mRNA concentrations by 57%. Its protein product interacts with calmodulin and calcium channel proteins. MBG treatment downregulated several genes whose protein products are involved in cell adhesion (ITGA2B, FERMT1, CLDN16, and TMEM207) and cell signaling (GRIN2C, SLC8A1, and ESR1). The level of downregulation ranged from 22 to 66%. Altogether, MBG actively enhanced the permeability of HBMEC monolayers while downregulating genes involved in adhesion. MBG treatment had variable effects on ENKUR, GRIN2C, and SLC8A1 genes, all associated with calcium transport. These studies provide the basis for future investigations of MBG actions in normal physiology and disease.

Angiotensin II receptor type 1--a novel target for preventing neonatal meningitis in mice by Escherichia coli K1.

The increasing incidence of Escherichia coli K1 meningitis due to escalating antibiotic resistance warrants alternate treatment options to prevent this deadly disease. We screened a library of small molecules from the National Institutes of Health clinical collection and identified telmisartan, an angiotensin II receptor type 1 (AT1R) blocker, as a potent inhibitor of E. coli invasion into human brain microvascular endothelial cells (HBMECs). Immunoprecipitation studies revealed that AT1R associates with endothelial cell gp96, the receptor in HBMECs for E. coli outer membrane protein A. HBMECs pretreated with telmisartan or transfected with AT1R small interfering RNA were resistant to E. coli invasion because of downregulation of protein kinase C-α phosphorylation. Administration of a soluble derivative of telmisartan to newborn mice before infection with E. coli prevented the onset of meningitis and suppressed neutrophil infiltration and glial cell migration in the brain. Therefore, telmisartan has potential as an alternate treatment option for preventing E. coli meningitis.