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

2014 ◽  
Vol 145 ◽  
pp. S78-S83 ◽  
Author(s):  
Junaid Iqbal ◽  
Komal Naeem ◽  
Ruqaiyyah Siddiqui ◽  
Naveed Ahmed Khan
Keyword(s):  
Endothelial Cells ◽  
Human Brain ◽  
Acanthamoeba Castellanii ◽  
Microvascular Endothelial Cells ◽  
Brain Microvascular Endothelial Cells ◽  
Protease Activated Receptors ◽  
In Vitro Inhibition ◽  
Microvascular Endothelial

scholarly journals Single Domain Antibodies Targeting Receptor Binding Pockets of NadA Restrain Adhesion of Neisseria meningitidis to Human Brain Microvascular Endothelial Cells

2020 ◽  
Vol 7 ◽  
Author(s):  
Amod Kulkarni ◽  
Evelína Mochnáčová ◽  
Petra Majerova ◽  
Ján Čurlík ◽  
Katarína Bhide ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Phage Display ◽  
Human Brain ◽  
Receptor Binding ◽  
Phage Library ◽  
Microvascular Endothelial Cells ◽  
Brain Microvascular Endothelial Cells ◽  
Binding Pockets ◽  
Microvascular Endothelial

Neisseria adhesin A (NadA), one of the surface adhesins of Neisseria meningitides (NM), interacts with several cell types including human brain microvascular endothelial cells (hBMECs) and play important role in the pathogenesis. Receptor binding pockets of NadA are localized on the globular head domain (A33 to K69) and the first coiled-coil domain (L121 to K158). Here, the phage display was used to develop a variable heavy chain domain (VHH) that can block receptor binding sites of recombinant NadA (rec-NadA). A phage library displaying VHH was panned against synthetic peptides (NadA-gdA33−K69 or NadA-ccL121−K158), gene encoding VHH was amplified from bound phages and re-cloned in the expression vector, and the soluble VHHs containing disulfide bonds were overexpressed in the SHuffle E. coli. From the repertoire of 96 clones, two VHHs (VHHF3–binding NadA-gdA33−K69 and VHHG9–binding NadA-ccL121−K158) were finally selected as they abrogated the interaction between rec-NadA and the cell receptor. Preincubation of NM with VHHF3 and VHHG9 significantly reduced the adhesion of NM on hBMECs in situ and hindered the traversal of NM across the in-vitro BBB model. The work presents a phage display pipeline with a single-round of panning to select receptor blocking VHHs. It also demonstrates the production of soluble and functional VHHs, which blocked the interaction between NadA and its receptor, decreased adhesion of NM on hBMECs, and reduced translocation of NM across BBB in-vitro. The selected NadA blocking VHHs could be promising molecules for therapeutic translation.

Differential Ca2+signaling by thrombin and protease-activated receptor-1-activating peptide in human brain microvascular endothelial cells

2004 ◽  
Vol 286 (1) ◽  
pp. C31-C42 ◽  
Author(s):  
Yuri V. Kim ◽  
Francescopaolo Di Cello ◽  
Coryse S. Hillaire ◽  
Kwang Sik Kim
Keyword(s):  
Endothelial Cells ◽  
Human Brain ◽  
Microvascular Endothelial Cells ◽  
Brain Microvascular Endothelial Cells ◽  
Atpase Inhibitor ◽  
Protease Activated Receptors ◽  
Plasmic Reticulum ◽  
Intracellular Ca ◽  
Microvascular Endothelial ◽  
Stimulation Of

Thrombin and related protease-activated receptors 1, 2, 3, and 4 (PAR1–4) play a multifunctional role in many types of cells including endothelial cells. Here, using RT-PCR and immunofluorescence staining, we showed for the first time that PAR1–4 are expressed on primary human brain microvascular endothelial cells (HBMEC). Digital fluorescence microscopy and fura 2 were used to monitor intracellular Ca2+concentration ([Ca2+]i) changes in response to thrombin and PAR1-activating peptide (PAR1-AP) SFFLRN. Both thrombin and PAR1-AP induced a dose-dependent [Ca2+]irise that was inhibited by pretreatment of HBMEC with the phospholipase C inhibitor U-73122 and the sarco(endo)plasmic reticulum Ca2+-ATPase inhibitor thapsigargin. Thrombin induced transient [Ca2+]iincrease, whereas PAR1-AP exhibited sustained [Ca2+]irise. The PAR1-AP-induced sustained [Ca2+]irise was significantly reduced in the absence of extracellular calcium or in the presence of an inhibitor of store-operated calcium channels, SKF-96365. Restoration of extracellular Ca2+to the cells that were initially activated by PAR1-AP in the absence of extracellular Ca2+resulted in significant [Ca2+]irise; however, this effect was not observed after thrombin stimulation. Pretreatment of the cells with a low thrombin concentration (0.1 nM) prevented [Ca2+]irise in response to high thrombin concentration (10 nM), but pretreatment with PAR1-AP did not prevent subsequent [Ca2+]irise to high PAR1-AP concentration. Additionally, treatment with thrombin decreased transendothelial electrical resistance in HBMEC, whereas PAR1-AP was without significant effect. These findings suggest that, in contrast to thrombin, stimulation of PAR1 by untethered peptide SFFLRN results in stimulation of store-operated Ca2+influx without significantly affecting brain endothelial barrier functions.

scholarly journals Pinocembrin Protects Human Brain Microvascular Endothelial Cells against Fibrillar Amyloid-β1−40Injury by Suppressing the MAPK/NF-κB Inflammatory Pathways

2014 ◽  
Vol 2014 ◽  
pp. 1-14 ◽  
Author(s):  
Rui Liu ◽  
Jin-ze Li ◽  
Jun-ke Song ◽  
Jia-lin Sun ◽  
Yong-jie Li ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Human Brain ◽  
Nuclear Translocation ◽  
Microvascular Endothelial Cells ◽  
Therapeutic Effects ◽  
Brain Microvascular Endothelial Cells ◽  
Nuclear Condensation ◽  
Microvascular Endothelial ◽  
Anti Inflammation

Cerebrovascular accumulation of amyloid-β(Aβ) peptides in Alzheimer’s disease (AD) may contribute to disease progression through Aβ-induced microvascular endothelial pathogenesis. Pinocembrin has been shown to have therapeutic effects in AD models. These effects correlate with preservation of microvascular function, but the effect on endothelial cells under Aβ-damaged conditions is unclear. The present study focuses on thein vitroprotective effect of pinocembrin on fibrillar Aβ1−40(fAβ1−40) injured human brain microvascular endothelial cells (hBMECs) and explores potential mechanisms. The results demonstrate that fAβ1−40-induced cytotoxicity in hBMECs can be rescued by pinocembrin treatment. Pinocembrin increases cell viability, reduces the release of LDH, and relieves nuclear condensation. The mechanisms of this reversal from Aβmay be associated with the inhibition of inflammatory response, involving inhibition of MAPK activation, downregulation of phosphor-IKK level, relief of IκBαdegradation, blockage of NF-κB p65 nuclear translocation, and reduction of the release of proinflammatory cytokines. Pinocembrin does not show obvious effects on regulating the redox imbalance after exposure to fAβ1−40. Together, the suppression of MAPK and the NF-κB signaling pathways play a significant role in the anti-inflammation of pinocembrin in hBMECs subjected to fAβ1−40. This may serve as a therapeutic agent for BMEC protection in Alzheimer’s-related deficits.

scholarly journals miR-24 targets SARS-CoV-2 co-factor Neuropilin-1 in human brain microvascular endothelial cells: Insights for COVID-19 neurological manifestations

2021 ◽  
Author(s):  
Pasquale Mone ◽  
Jessica Gambardella ◽  
Xujun Wang ◽  
Stanislovas S. Jankauskas ◽  
Alessandro Matarese ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Human Brain ◽  
Blood Brain Barrier ◽  
Brain Barrier ◽  
Microvascular Endothelial Cells ◽  
Brain Microvascular Endothelial Cells ◽  
Blood Brain ◽  
Neuropilin 1 ◽  
Microvascular Endothelial

Abstract Neuropilin-1 is a transmembrane glycoprotein that has been implicated in several processes including angiogenesis and immunity. Recent evidence has also shown that it is implied in the cellular internalization of the severe acute respiratory syndrome coronavirus (SARS-CoV-2), which causes the coronavirus disease 2019 (COVID-19). We hypothesized that specific microRNAs can target Neuropilin-1. By combining bioinformatic and functional approaches, we identified miR-24 as a regulator of Neuropilin-1 transcription. Since Neuropilin-1 has been shown to play a key role in the endothelium-mediated regulation of the blood-brain barrier, we validated miR-24 as a functional modulator of Neuropilin-1 in human brain microvascular endothelial cells (hBMECs), which are the most suitable cell line for an in vitro blood–brain barrier model.

Regulation of Integrin Expression and Activation by VEGF in Human Brain Microvascular Endothelial Cells: Implication of α6 Integrin in Angiogenesis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2609-2609
Author(s):  
Tae-Hee Lee ◽  
Hava Karsenty Avraham ◽  
Huchun Li ◽  
Stephen J. Kennel ◽  
Shalom Avraham
Keyword(s):  
Endothelial Cells ◽  
Human Brain ◽  
Cell Surface Expression ◽  
Microvascular Endothelial Cells ◽  
Brain Microvascular Endothelial Cells ◽  
Rnase Protection ◽  
Microvascular Endothelial ◽  
And Migration

Abstract The precise role of vascular endothelial growth factor (VEGF) in the regulation of integrins is not well elucidated due to their high redundancy. Here, we examined the effects of VEGF on the expression and activation of integrins in human brain microvascular endothelial cells (HBMECs). Using human cDNA arrays and Ribonuclease (RNase) protection assays which cover most of the known integrins, we observed that VEGF significantly upregulated the mRNA expression of α1, α2, and α6 integrins in HBMECs. While VEGF was reported to induce α1 and α2 integrins, the observation of α6 integrin induction by VEGF is novel. Using small interfering RNA (siRNA) oligonucleotides for α6 integrin, we observed downregulation of the cell surface expression of α6 integrin in HBMECs. This downregulation resulted in inhibition of both HBMEC capillary morphogenesis and of the VEGF-induced adhesion and migration of the cells. VEGF also induced the activation of α6 integrin in the HBMECs. Functional blocking of α6 integrin by its specific antibody led to inhibition of VEGF-induced adhesion and migration as well as of in vivo angiogenesis, and significantly suppressed tumor angiogenesis and breast carcinoma cell growth in vivo. These results indicate that VEGF can modulate the in vitro angiogenesis of HBMECs via increased expression and activation of the α6 integrin, and that this integrin participates in VEGF-driven angiogenesis in vivo.

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