The induction of apoptosis in HLECs or HBECs co-cultured with <i>P. falciparum</i> PRBCs depends on the culture conditions.

<p>A) Co-cultured endothelial cells (ECs) and parasitized red blood cells (PRBCs) after Annexin V and propidium iodide (PI) staining. B) Human lung endothelial cell (HLEC) apoptosis induced by the different <i>P. falciparum</i> isolates (A, B and C) when the co-cultures are made in the standard culture media (LCm) or in the modified culture medium (MLCm). C) Human brain endothelial cell (HBEC) apoptosis induced by the different <i>P. falciparum</i> isolates (A, B and C) when the co-cultures are made in the standard culture media (BCm) or in the modified culture medium (MBCm). The percentage of apoptotic cells was calculated after microscopy enumeration of Annexin V and propidium iodide positive cells in 1000 ECs. The results are expressed as mean values, and standard deviation are shown for duplicate wells from three biological replicates. Significance at <i>p</i><0.005 is indicated by **.</p

Primers used for reverse-transcriptase polymerase chain reaction (RT-qPCR) experiments.

<p>Primers used for reverse-transcriptase polymerase chain reaction (RT-qPCR) experiments.</p

Direct cell-to-cell contact is needed for optimal induction of EC apoptosis.

<p>HLEC (A) and HBEC (B) co-cultured with individual supernatants harvested from cultured <i>P. falciparum</i> isolates or in transwell plates where the PRBC are incubated on top of the ECs. The results show reduced levels of apoptosis as compared to those observed in co-cultures where PRBC and ECs are in direct contact. For all experiments the means and standard deviation were calculated from duplicate wells from three biological replicates. Significance at <i>p</i><0.05 is indicated by *.</p

Variation of gene expression when <i>P. falciparum</i> isolates were incubated with ECs.

<p>The implication of the seven selected genes in the ability of isolates B and C to induce the lung and brain EC death was represented as a ratio by comparing their RNA levels in co-cultures using the modified culture mediums, where EC apoptosis death was detected. For all experiments the means and standard deviation were calculated from duplicate wells from two biological replicates.</p><p>*p<0.05,</p><p>**p<0.005,</p><p>***p<0.0005.</p><p>NS = Non Significant.</p

Differences in amyloid-β clearance across mouse and human blood-brain barrier models: kinetic analysis and mechanistic modeling.

Alzheimer's disease (AD) has a characteristic hallmark of amyloid-β (Aβ) accumulation in the brain. This accumulation of Aβ has been related to its faulty cerebral clearance. Indeed, preclinical studies that used mice to investigate Aβ clearance showed that efflux across blood-brain barrier (BBB) and brain degradation mediate efficient Aβ clearance. However, the contribution of each process to Aβ clearance remains unclear. Moreover, it is still uncertain how species differences between mouse and human could affect Aβ clearance. Here, a modified form of the brain efflux index method was used to estimate the contribution of BBB and brain degradation to Aβ clearance from the brain of wild type mice. We estimated that 62% of intracerebrally injected 125I-Aβ40 is cleared across BBB while 38% is cleared by brain degradation. Furthermore, in vitro and in silico studies were performed to compare Aβ clearance between mouse and human BBB models. Kinetic studies for Aβ40 disposition in bEnd3 and hCMEC/D3 cells, representative in vitro mouse and human BBB models, respectively, demonstrated 30-fold higher rate of 125I-Aβ40 uptake and 15-fold higher rate of degradation by bEnd3 compared to hCMEC/D3 cells. Expression studies showed both cells to express different levels of P-glycoprotein and RAGE, while LRP1 levels were comparable. Finally, we established a mechanistic model, which could successfully predict cellular levels of 125I-Aβ40 and the rate of each process. Established mechanistic model suggested significantly higher rates of Aβ uptake and degradation in bEnd3 cells as rationale for the observed differences in 125I-Aβ40 disposition between mouse and human BBB models. In conclusion, current study demonstrates the important role of BBB in the clearance of Aβ from the brain. Moreover, it provides insight into the differences between mouse and human BBB with regards to Aβ clearance and offer, for the first time, a mathematical model that describes Aβ clearance across BBB

Expression of inflammatory genes induced by beta-amyloid peptides in human brain endothelial cells and in Alzheimer's brain is mediated by the JNK-AP1 signaling pathway

Alzheimer's disease (AD) is characterized by accumulation and deposition of Abeta peptides in the brain. Abeta deposition in cerebral vessels occurs in many AD patients and results in cerebral amyloid angiopathy (AD/CAA). Abeta deposits evoke neuro- and neurovascular inflammation contributing to neurodegeneration. In this study, we found that exposure of cultured human brain endothelial cells (HBEC) to Abeta(1-40) elicited expression of inflammatory genes MCP-1, GRO, IL-1beta and IL-6. Up-regulation of these genes was confirmed in AD and AD/CAA brains by qRT-PCR. Profiling of 54 transcription factors indicated that AP-1 was strongly activated not only in Abeta-treated HBEC but also in AD and AD/CAA brains. AP-1 complex in nuclear extracts from Abeta-treated HBEC bound to AP-1 DNA-binding sequence and activated the reporter gene of a luciferase vector carrying AP-1-binding site from human MCP-1 gene. AP-1 is a dimeric protein complex and supershift assay identified c-Jun as a component of the activated AP-1 complex. Western blot analyses showed that c-Jun was activated via JNK-mediated phosphorylation, suggesting that as a result of c-Jun phosphorylation, AP-1 was activated and thus up-regulated MCP-1 expression. A JNK inhibitor SP600125 strongly inhibited Abeta-induced c-Jun phosphorylation, AP-1 activation, AP-1 reporter gene activity and MCP-1 expression in cells stimulated with Abeta peptides. The results suggested that JNK-AP1 signaling pathway is responsible for Abeta-induced neuroinflammation in HBEC and Alzheimer's brain and that this signaling pathway may serve as a therapeutic target for relieving Abeta-induced inflammationNRC publication: Ye

P. falciparum Isolate-Specific Distinct Patterns of Induced Apoptosis in Pulmonary and Brain Endothelial Cells

International audienceThe factors implicated in the transition from uncomplicated to severe clinical malaria such as pulmonary oedema and cerebral malaria remain unclear. It is known that alterations in vascular integrity due to endothelial cell (EC) activation and death occur during severe malaria. In this study, we assessed the ability of different P. falciparum clinical isolates to induce apoptosis in ECs derived from human lung and brain. We observed that induction of EC apoptosis was sensitive to the environmental pH and required direct contact between the parasite and the cell, though it was not correlated to the ability of the parasite to cytoadhere. Moreover, the extent of induced apoptosis in the two EC types varied with the isolate. Analysis of parasite genes transcript led us to propose that the activation of different pathways, such as Plasmodium apoptosis–linked pathogenicity factors (PALPF), PALPF-2, PALPF-5 and PF11_0521, could be implied in EC death. These observations provide an experimental framework to decipher the molecular mechanism implicated in the genesis of severe malaria

Cilostazol strengthens barrier integrity in brain endothelial cells

We studied the effect of cilostazol, a selective inhibitor of phosphodiesterase 3, on barrier functions of blood–brain barrier (BBB)-related endothelial cells, primary rat brain capillary endothelial cells (RBEC), and the immortalized human brain endothelial cell line hCMEC/D3. The pharmacological potency of cilostazol was also evaluated on ischemia-related BBB dysfunction using a triple co-culture BBB model (BBB Kit™) subjected to 6-h oxygen glucose deprivation (OGD) and 3-h reoxygenation. There was expression of phosphodiesterase 3B mRNA in RBEC, and a significant increase in intracellular cyclic AMP (cAMP) content was detected in RBEC treated with both 1 and 10 μM cilostazol. Cilostazol increased the transendothelial electrical resistance (TEER), an index of barrier tightness of interendothelial tight junctions (TJs), and decreased the endothelial permeability of sodium fluorescein through the RBEC monolayer. The effects on these barrier functions were significantly reduced in the presence of protein kinase A (PKA) inhibitor H-89. Microscopic observation revealed smooth and even localization of occludin immunostaining at TJs and F-actin fibers at the cell borders in cilostazol-treated RBEC. In hCMEC/D3 cells treated with 1 and 10 μM cilostazol for 24 and 96 h, P-glycoprotein transporter activity was increased, as assessed by rhodamine 123 accumulation. Cilostazol improved the TEER in our triple co-culture BBB model with 6-h OGD and 3-h reoxygenation. As cilostazol stabilized barrier integrity in BBB-related endothelial cells, probably via cAMP/PKA signaling, the possibility that cilostazol acts as a BBB-protective drug against cerebral ischemic insults to neurons has to be considered