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2022 ◽  
Vol 17 (1) ◽  
Author(s):  
Jun Wang ◽  
Dong-Yu Fan ◽  
Hui-Yun Li ◽  
Chen-Yang He ◽  
Ying-Ying Shen ◽  
...  

Abstract Background Loss of brain capillary pericyte is involved in the pathologies and cognitive deficits in Alzheimer’s disease (AD). The role of pericyte in early stage of AD pathogenesis remains unclear. Methods We investigated the dynamic changes of soluble platelet-derived growth factor receptor β (sPDGFRβ) in cerebrospinal fluid (CSF), a marker of brain pericyte injury, in transition from normal ageing to early AD in a cognitively unimpaired population aged 20 to 90 years. Association between sPDGFRβ and ATN biomarkers were analyzed. Results In lifetime, CSF sPDGFRβ continually increased since age of 20 years, followed by the increases of phosphorylated tau-181 (P-tau181) and total tau (T-tau) at the age of 22.2 years and 31.7 years, respectively; CSF Aβ42 began to decline since the age of 39.6 years, indicating Aβ deposition. The natural trajectories of biomarkers suggest that pericyte injury is an early event during transition from normal status to AD, even earlier than Aβ deposition. In AD spectrum, CSF sPDGFRβ was elevated in preclinical stage 2 and participants with suspected non-AD pathophysiologies. Additionally, CSF sPDGFRβ was positively associated with P-tau181 and T-tau independently of Aβ42, and significantly strengthened the effects of Aβ42 on P-tau181, suggesting that pericyte injury accelerates Aβ-mediated tau hyperphosphorylation. Conclusions Our results suggest that pericyte injury contributes to AD progression in the early stage in an Aβ-independent pathway. Recovery of pericyte function would be a target for prevention and early intervention of AD.


2022 ◽  
Vol 23 (2) ◽  
pp. 742
Author(s):  
Shireen Mentor ◽  
Khayelihle Brian Makhathini ◽  
David Fisher

The brain capillary endothelium is highly regulatory, maintaining the chemical stability of the brain’s microenvironment. The role of cytoskeletal proteins in tethering nanotubules (TENTs) during barrier-genesis was investigated using the established immortalized mouse brain endothelial cell line (bEnd5) as an in vitro blood-brain barrier (BBB) model. The morphology of bEnd5 cells was evaluated using both high-resolution scanning electron microscopy and immunofluorescence to evaluate treatment with depolymerizing agents Cytochalasin D for F-actin filaments and Nocodazole for α-tubulin microtubules. The effects of the depolymerizing agents were investigated on bEnd5 monolayer permeability by measuring the transendothelial electrical resistance (TEER). The data endorsed that during barrier-genesis, F-actin and α-tubulin play a cytoarchitectural role in providing both cell shape dynamics and cytoskeletal structure to TENTs forming across the paracellular space to provide cell-cell engagement. Western blot analysis of the treatments suggested a reduced expression of both proteins, coinciding with a reduction in the rates of cellular proliferation and decreased TEER. The findings endorsed that TENTs provide alignment of the paracellular (PC) spaces and tight junction (TJ) zones to occlude bEnd5 PC spaces. The identification of specific cytoskeletal structures in TENTs endorsed the postulate of their indispensable role in barrier-genesis and the maintenance of regulatory permeability across the BBB.


2022 ◽  
Vol 19 (1) ◽  
Author(s):  
Burak Ozgür ◽  
Hans Christian Cederberg Helms ◽  
Erica Tornabene ◽  
Birger Brodin

Abstract Background Brain capillary endothelial cells (BCECs) experience hypoxic conditions during early brain development. The newly formed capillaries are tight and functional before astrocytes and pericytes join the capillaries and establish the neurovascular unit. Brain endothelial cell phenotype markers P-gp (ABCB1), LAT-1(SLC7A5), GLUT-1(SLC2A1), and TFR(TFRC) have all been described to be hypoxia sensitive. Therefore, we hypothesized that monolayers of BCECs, cultured under hypoxic conditions, would show an increase in LAT-1, GLUT-1 and TFR expression and display tight endothelial barriers. Methods and results Primary bovine BCECs were cultured under normoxic and hypoxic conditions. Chronic hypoxia induced HIF-1α stabilization and translocation to the nucleus, as judged by immunocytochemistry and confocal laser scanning imaging. Endothelial cell morphology, claudin-5 and ZO-1 localization and barrier integrity were unaffected by hypoxia, indicating that the tight junctions in the BBB model were not compromised. SLC7A5, SLC2A1, and TFRC-mRNA levels were increased in hypoxic cultures, while ABCB1 remained unchanged as shown by real-time qPCR. P-gp, TfR and GLUT-1 were found to be significantly increased at protein levels. An increase in uptake of [3H]-glucose was demonstrated, while a non-significant increase in the efflux ratio of the P-gp substrate [3H]-digoxin was observed in hypoxic cells. No changes were observed in functional LAT-1 as judged by uptake studies of [3H]-leucine. Stabilization of HIF-1α under normoxic conditions with desferrioxamine (DFO) mimicked the effects of hypoxia on endothelial cells. Furthermore, low concentrations of DFO caused an increase in transendothelial electrical resistance (TEER), suggesting that a slight activation of the HIF-1α system may actually increase brain endothelial monolayer tightness. Moreover, exposure of confluent monolayers to hypoxia resulted in markedly increase in TEER after 24 and 48 h, which corresponded to a higher transcript level of CLDN5. Conclusions Our findings collectively suggest that hypoxic conditions increase some BBB transporters' expression via HIF-1α stabilization, without compromising monolayer integrity. This may in part explain why brain capillaries show early maturation, in terms of barrier tightness and protein expression, during embryogenesis, and provides a novel methodological tool for optimal brain endothelial culture.


Pathogens ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 1598
Author(s):  
Naomi S. Taus ◽  
Colette Cywes-Bentley ◽  
Wendell C. Johnson ◽  
Gerald B. Pier ◽  
Lindsay M. Fry ◽  
...  

Arthropod-borne apicomplexan pathogens remain a great concern and challenge for disease control in animals and humans. In order to prevent Babesia infection, the discovery of antigens that elicit protective immunity is essential to establish approaches to stop disease dissemination. In this study, we determined that poly-N-acetylglucosamine (PNAG) is conserved among tick-borne pathogens including B. bovis, B. bigemina, B. divergens, B. microti, and Babesia WA1. Calves immunized with synthetic ß-(1→6)-linked glucosamine oligosaccharides conjugated to tetanus toxoid (5GlcNH2-TT) developed antibodies with in vitro opsonophagocytic activity against Staphylococcus aureus. Sera from immunized calves reacted to B. bovis. These results suggest strong immune responses against PNAG. However, 5GlcNH2-TT-immunized bovines challenged with B. bovis developed acute babesiosis with the cytoadhesion of infected erythrocytes to brain capillary vessels. While this antigen elicited antibodies that did not prevent disease, we are continuing to explore other antigens that may mitigate these vector-borne diseases for the cattle industry.


2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Birthe Gericke ◽  
Saskia Borsdorf ◽  
Inka Wienböker ◽  
Andreas Noack ◽  
Sandra Noack ◽  
...  

Abstract Background In vitro models based on brain capillary endothelial cells (BCECs) are among the most versatile tools in blood–brain barrier research for testing drug penetration into the brain and how this is affected by efflux transporters such as P-glycoprotein (Pgp). However, compared to freshly isolated brain capillaries or primary BCECs, the expression of Pgp in immortalized BCEC lines is markedly lower, which prompted us previously to transduce the widely used human BCEC line hCMEC/D3 with a doxycycline-inducible MDR1-EGFP fusion plasmid. The EGFP-labeled Pgp in these cells allows studying the localization and trafficking of the transporter and how these processes are affected by drug exposure. Here we used this strategy for the rat BCEC line RBE4 and performed a face-to-face comparison of RBE4 and hCMEC/D3 wild-type (WT) and MDR1-EGFP transduced cells. Methods MDR1-EGFP-transduced variants were derived from WT cells by lentiviral transduction, using an MDR1-linker-EGFP vector. Localization, trafficking, and function of Pgp were compared in WT and MDR1-EGFP transduced cell lines. Primary cultures of rat BCECs and freshly isolated rat brain capillaries were used for comparison. Results All cells exhibited typical BCEC morphology. However, significant differences were observed in the localization of Pgp in that RBE4-MDR1-EGFP cells expressed Pgp primarily at the plasma membrane, whereas in hCMEC/D3 cells, the Pgp-EGFP fusion protein was visible both at the plasma membrane and in endolysosomal vesicles. Exposure to doxorubicin increased the number of Pgp-EGFP-positive endolysosomes, indicating a lysosomotropic effect. Furthermore, lysosomal trapping of doxorubicin was observed, likely contributing to the protection of the cell nucleus from damage. In cocultures of WT and MDR1-EGFP transduced cells, intercellular Pgp-EGFP trafficking was observed in RBE4 cells as previously reported for hCMEC/D3 cells. Compared to WT cells, the MDR1-EGFP transduced cells exhibited a significantly higher expression and function of Pgp. However, the junctional tightness of WT and MDR1-EGFP transduced RBE4 and hCMEC/D3 cells was markedly lower than that of primary BCECs, excluding the use of the cell lines for studying vectorial drug transport. Conclusions The present data indicate that MDR1-EGFP transduced RBE4 cells are an interesting tool to study the biogenesis of lysosomes and Pgp-mediated lysosomal drug trapping in response to chemotherapeutic agents and other compounds at the level of the blood–brain barrier.


2021 ◽  
Vol 7 (30) ◽  
pp. eabh0101
Author(s):  
Thomas A. Longden ◽  
Amreen Mughal ◽  
Grant W. Hennig ◽  
Osama F. Harraz ◽  
Bo Shui ◽  
...  

Healthy brain function depends on the finely tuned spatial and temporal delivery of blood-borne nutrients to active neurons via the vast, dense capillary network. Here, using in vivo imaging in anesthetized mice, we reveal that brain capillary endothelial cells control blood flow through a hierarchy of IP3 receptor–mediated Ca2+ events, ranging from small, subsecond protoevents, reflecting Ca2+ release through a small number of channels, to high-amplitude, sustained (up to ~1 min) compound events mediated by large clusters of channels. These frequent (~5000 events/s per microliter of cortex) Ca2+ signals are driven by neuronal activity, which engages Gq protein–coupled receptor signaling, and are enhanced by Ca2+ entry through TRPV4 channels. The resulting Ca2+-dependent synthesis of nitric oxide increases local blood flow selectively through affected capillary branches, providing a mechanism for high-resolution control of blood flow to small clusters of neurons.


2021 ◽  
Vol 118 (26) ◽  
pp. e2100866118
Author(s):  
Vanessa Coelho-Santos ◽  
Andrée-Anne Berthiaume ◽  
Sharon Ornelas ◽  
Heidi Stuhlmann ◽  
Andy Y. Shih

Capillary networks are essential for distribution of blood flow through the brain, and numerous other homeostatic functions, including neurovascular signal conduction and blood–brain barrier integrity. Accordingly, the impairment of capillary architecture and function lies at the root of many brain diseases. Visualizing how brain capillary networks develop in vivo can reveal innate programs for cerebrovascular growth and repair. Here, we use longitudinal two-photon imaging through noninvasive thinned skull windows to study a burst of angiogenic activity during cerebrovascular development in mouse neonates. We find that angiogenesis leading to the formation of capillary networks originated exclusively from cortical ascending venules. Two angiogenic sprouting activities were observed: 1) early, long-range sprouts that directly connected venules to upstream arteriolar input, establishing the backbone of the capillary bed, and 2) short-range sprouts that contributed to expansion of anastomotic connectivity within the capillary bed. All nascent sprouts were prefabricated with an intact endothelial lumen and pericyte coverage, ensuring their immediate perfusion and stability upon connection to their target vessels. The bulk of this capillary expansion spanned only 2 to 3 d and contributed to an increase of blood flow during a critical period in cortical development.


2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Rahul Basu ◽  
Vinod Nair ◽  
Clayton W. Winkler ◽  
Tyson A. Woods ◽  
Iain D. C. Fraser ◽  
...  

Abstract Background A key factor in the development of viral encephalitis is a virus crossing the blood-brain barrier (BBB). We have previously shown that age-related susceptibility of mice to the La Crosse virus (LACV), the leading cause of pediatric arbovirus encephalitis in the USA, was associated with the ability of the virus to cross the BBB. LACV infection in weanling mice (aged around 3 weeks) results in vascular leakage in the olfactory bulb/tract (OB/OT) region of the brain, which is not observed in adult mice aged > 6–8 weeks. Thus, we studied age-specific differences in the response of brain capillary endothelial cells (BCECs) to LACV infection. Methods To examine mechanisms of LACV-induced BBB breakdown and infection of the CNS, we analyzed BCECs directly isolated from weanling and adult mice as well as established a model where these cells were infected in vitro and cultured for a short period to determine susceptibility to virus infection and cell death. Additionally, we utilized correlative light electron microscopy (CLEM) to examine whether changes in cell morphology and function were also observed in BCECs in vivo. Results BCECs from weanling, but not adult mice, had detectable infection after several days in culture when taken ex vivo from infected mice suggesting that these cells could be infected in vitro. Further analysis of BCECs from uninfected mice, infected in vitro, showed that weanling BCECs were more susceptible to virus infection than adult BCECs, with higher levels of infected cells, released virus as well as cytopathic effects (CPE) and cell death. Although direct LACV infection is not detected in the weanling BCECs, CLEM analysis of brain tissue from weanling mice indicated that LACV infection induced significant cerebrovascular damage which allowed virus-sized particles to enter the brain parenchyma. Conclusions These findings indicate that BCECs isolated from adult and weanling mice have differential viral load, infectivity, and susceptibility to LACV. These age-related differences in susceptibility may strongly influence LACV-induced BBB leakage and neurovascular damage allowing virus invasion of the CNS and the development of neurological disease.


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