Abstract TP276: Perlecan Is Required for the Maintenance of the Blood-Brain Barrier through the Interaction with Pericytes in a Mouse Ischemic Stroke Model

Stroke ◽  
2017 ◽  
Vol 48 (suppl_1) ◽  
Author(s):  
Kuniyuki Nakamura ◽  
Tomoko Ikeuchi ◽  
Peipei Zhang ◽  
Craig Rhodes ◽  
Yuta Chiba ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Ischemic Stroke ◽  
Endothelial Cell ◽  
Blood Brain Barrier ◽  
Repair Process ◽  
Ischemic Lesion ◽  
Brain Vasculature ◽  
Integrin Α5 ◽  
The Brain

Introduction: The disruption of the blood-brain barrier (BBB) is a contributing factor for the deterioration of brain damages in ischemic stroke. Recent studies revealed that microvascular pericytes are involved in the maintenance of the BBB and in the repair process through platelet-derived growth factor receptor β (PDGFRβ), the expression of which is increased around the ischemic lesion. Here we focus on perlecan, the major heparan sulfate proteoglycan in the basement membrane (BM). It is expressed by endothelial cells in the brain and is implicated in many biological functions. In this report, we have studied the role of Perlecan in the BBB breakdown and in the subsequent repair process after ischemic stroke in a mouse model. We hypothesized that perlecan may play a protective role in the disruption of BBB through the interaction with pericytes after ischemic stroke. Methods: To elucidate the role of perlecan in the brain vasculature, we induced a 60-minute transient middle cerebral artery occlusion (MCAO) in adult conditional perlecan -deficient ( Perlecan -/- -Tg) mice, which express the perlecan transgene only in the cartilage to rescue the perinatal lethality of perlecan -deficient mice. Results: Although the BBB formation and function in the brain vasculature appeared to be unaffected in Perlecan -/- -Tg mice under healthy condition, Perlecan -/- -Tg mice demonstrated larger infarct volumes and more BBB leakage than control mice on post-surgery day (PSD) 2 after MCAO. Perlecan -/- -Tg mice exhibited less PDGFRβ-positive pericytes around the ischemic lesion on PSD 3 to 7 than control mice, suggesting that the perlecan deficiency suppressed pericyte activation. At a mechanistic level, integrin α5, a potential receptor for perlecan, was detectable in both endothelial cells and pericytes in the ischemic lesion, suggesting that endothelial cell-derived perlecan may regulate pericyte activation in response to ischemia through integrin α5. Conclusions: Our results suggest that perlecan is required for the activation of pericytes and thereby, contributing to the endothelial cell-pericyte integrity in the BBB maintenance after ischemic stroke.

scholarly journals Characterization and tissue localization of zebrafish homologs of the human ABCB1 multidrug transporter

2021 ◽  
Author(s):  
Robert W. Robey ◽  
Andrea N. Robinson ◽  
Fatima Ali-Rahmani ◽  
Lyn M. Huff ◽  
Sabrina Lusvarghi ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Multidrug Transporter ◽  
Glycoprotein P ◽  
Tissue Localization ◽  
Brain Vasculature ◽  
P Glycoprotein ◽  
Capillary Endothelial Cells ◽  
The Brain ◽  
Viable Model

ABSTRACTGiven its similarities with mammalian systems, the zebrafish has emerged as a potential model to study the blood-brain barrier (BBB). Capillary endothelial cells at the human BBB express high levels of P-glycoprotein (P-gp, encoded by the ABCB1 gene) and ABCG2 (encoded by the ABCG2 gene). However, little information has been available about ATP-binding cassette transporters expressed at the zebrafish BBB. In this study, we focus on the characterization and tissue localization of two genes that are similar to human ABCB1, zebrafish abcb4 and abcb5. Cytotoxicity assays with stably-transfected cell lines revealed that zebrafish Abcb5 cannot efficiently transport the substrates doxorubicin and mitoxantrone compared to human P-gp and zebrafish Abcb4. Additionally, zebrafish Abcb5 did not transport the fluorescent probes BODIPY-ethylenediamine or LDS 751, while they were readily transported by Abcb4 and P-gp. A high-throughput screen conducted with 90 human P-gp substrates confirmed that zebrafish Abcb4 has overlapping substrate specificity with P-gp. Basal ATPase activity of zebrafish Abcb4 and Abcb5 was comparable to that of human P-gp. In the brain vasculature, RNAscope probes to detect abcb4 colocalized with staining by the P-gp antibody C219, while abcb5 was not detected. Zebrafish abcb4 also colocalized with claudin-5 expression in brain endothelial cells. Abcb4 and Abcb5 had different tissue localizations in multiple zebrafish tissues, consistent with different functions. The data suggest that zebrafish Abcb4 most closely phenocopies P-gp and that the zebrafish may be a viable model to study the role of the multidrug transporter P-gp at the BBB.

scholarly journals The Dual Role of Microglia in Blood-Brain Barrier Dysfunction after Stroke

2020 ◽  
Vol 18 (12) ◽  
pp. 1237-1249 ◽  
Author(s):  
Ruiqing Kang ◽  
Marcin Gamdzyk ◽  
Cameron Lenahan ◽  
Jiping Tang ◽  
Sheng Tan ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Blood Brain Barrier ◽  
Vital Role ◽  
Dual Role ◽  
Brain Barrier ◽  
Barrier Dysfunction ◽  
Blood Brain ◽  
M2 Microglia ◽  
The Brain

It is well-known that stroke is one of the leading causes of death and disability all over the world. After a stroke, the blood-brain barrier subsequently breaks down. The BBB consists of endothelial cells surrounded by astrocytes. Microglia, considered the long-living resident immune cells of the brain, play a vital role in BBB function. M1 microglia worsen BBB disruption, while M2 microglia assist in repairing BBB damage. Microglia can also directly interact with endothelial cells and affect BBB permeability. In this review, we are going to discuss the mechanisms responsible for the dual role of microglia in BBB dysfunction after stroke.

scholarly journals Perlecan regulates pericyte dynamics in the maintenance and repair of the blood–brain barrier

2019 ◽  
Vol 218 (10) ◽  
pp. 3506-3525 ◽  
Author(s):  
Kuniyuki Nakamura ◽  
Tomoko Ikeuchi ◽  
Kazuki Nara ◽  
Craig S. Rhodes ◽  
Peipei Zhang ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Blood Brain Barrier ◽  
Repair Process ◽  
Artery Occlusion ◽  
Basement Membranes ◽  
Brain Barrier ◽  
Integrin Α5β1 ◽  
Ischemic Lesion ◽  
Maintenance And Repair ◽  
Blood Brain

Ischemic stroke causes blood–brain barrier (BBB) breakdown due to significant damage to the integrity of BBB components. Recent studies have highlighted the importance of pericytes in the repair process of BBB functions triggered by PDGFRβ up-regulation. Here, we show that perlecan, a major heparan sulfate proteoglycan of basement membranes, aids in BBB maintenance and repair through pericyte interactions. Using a transient middle cerebral artery occlusion model, we found larger infarct volumes and more BBB leakage in conditional perlecan (Hspg2)-deficient (Hspg2−/−-TG) mice than in control mice. Control mice showed increased numbers of pericytes in the ischemic lesion, whereas Hspg2−/−-TG mice did not. At the mechanistic level, pericytes attached to recombinant perlecan C-terminal domain V (perlecan DV, endorepellin). Perlecan DV enhanced the PDGF-BB–induced phosphorylation of PDGFRβ, SHP-2, and FAK partially through integrin α5β1 and promoted pericyte migration. Perlecan therefore appears to regulate pericyte recruitment through the cooperative functioning of PDGFRβ and integrin α5β1 to support BBB maintenance and repair following ischemic stroke.

scholarly journals The secreted neuronal signal Spock1 regulates the blood-brain barrier

2021 ◽  
Author(s):  
Natasha M O'Brown ◽  
Nikit B Patel ◽  
Ursula Hartmann ◽  
Allon M Klein ◽  
Chenghua Gu ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Blood Brain Barrier ◽  
Single Layer ◽  
Brain Barrier ◽  
Cellular Interactions ◽  
Brain Vasculature ◽  
Blood Brain ◽  
Functional Development ◽  
Neuronal Signal ◽  
The Brain

The blood-brain barrier (BBB) is comprised of a single layer of endothelial cells with uniquely restrictive properties required for maintaining a tightly controlled homeostatic environment in the brain. Classic quail-chick grafting experiments showed that BBB properties are not intrinsic to brain endothelial cells, but instead are induced by signals from the embryonic brain microenvironment. Here we have identified a neuronally produced signal, Spock1, that specifically regulates BBB functional development in both zebrafish and mice without affecting angiogenesis. Using a combination of mosaic genetic analysis, tracer leakage assays and live imaging we show that Spock1 from neurons can regulate brain vasculature permeability non-cell autonomously. Electron microscopy analyses of spock1 mutants revealed that the leakage arises predominantly through increased endothelial transcytosis of both clathrin-independent small and large vesicles due to altered pericyte-endothelial interactions. Single-cell RNA sequencing analyses revealed a reduction in vascular expression of the cell adhesion molecule mcamb in the spock1 mutants, and this down-regulation of mcamb occurred specifically in regions with increased BBB leakage. These analyses indicate that the neuronal signal Spock1 regulates BBB properties by altering vascular gene expression and cellular interactions.

scholarly journals Characterization and tissue localization of zebrafish homologs of the human ABCB1 multidrug transporter

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Robert W. Robey ◽  
Andrea N. Robinson ◽  
Fatima Ali-Rahmani ◽  
Lyn M. Huff ◽  
Sabrina Lusvarghi ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
High Throughput Screening ◽  
Hek293 Cells ◽  
Glycoprotein P ◽  
Tissue Localization ◽  
Brain Vasculature ◽  
P Glycoprotein ◽  
Capillary Endothelial Cells ◽  
The Brain

AbstractCapillary endothelial cells of the human blood–brain barrier (BBB) express high levels of P-glycoprotein (P-gp, encoded by ABCB1) and ABCG2 (encoded by ABCG2). However, little information is available regarding ATP-binding cassette transporters expressed at the zebrafish BBB, which has emerged as a potential model system. We report the characterization and tissue localization of two genes that are similar to ABCB1, zebrafish abcb4 and abcb5. When stably expressed in HEK293 cells, both Abcb4 and Abcb5 conferred resistance to P-gp substrates; however, Abcb5 poorly transported doxorubicin and mitoxantrone compared to zebrafish Abcb4. Additionally, Abcb5 did not transport the fluorescent P-gp probes BODIPY-ethylenediamine or LDS 751, while they were transported by Abcb4. High-throughput screening of 90 human P-gp substrates confirmed that Abcb4 has an overlapping substrate specificity profile with P-gp. In the brain vasculature, RNAscope probes for abcb4 colocalized with staining by the P-gp antibody C219, while abcb5 was not detected. The abcb4 probe also colocalized with claudin-5 in brain endothelial cells. Abcb4 and Abcb5 had different tissue localizations in multiple zebrafish tissues, potentially indicating different functions. The data suggest that zebrafish Abcb4 functionally phenocopies P-gp and that the zebrafish may serve as a model to study the role of P-gp at the BBB.

scholarly journals Vasa Vasorum Lumen Narrowing in Brain Vascular Hyalinosis in Systemic Hypertension Patients Who Died of Ischemic Stroke

2020 ◽  
Vol 21 (24) ◽  
pp. 9611
Author(s):  
Sergiy G. Gychka ◽  
Nataliia V. Shults ◽  
Sofia I. Nikolaienko ◽  
Lucia Marcocci ◽  
Nurefsan E. Sariipek ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Ischemic Stroke ◽  
Endothelial Cell ◽  
Actin Polymerization ◽  
Vasa Vasorum ◽  
Postmortem Brain ◽  
Systemic Hypertension ◽  
Brain Vasculature ◽  
Cell Protrusion ◽  
Lumen Narrowing

Ischemic stroke is a major cause of death among patients with systemic hypertension. The narrowing of the lumen of the brain vasculature contributes to the increased incidence of stroke. While hyalinosis represents the major pathological lesions contributing to vascular lumen narrowing and stroke, the pathogenic mechanism of brain vascular hyalinosis has not been well characterized. Thus, the present study examined the postmortem brain vasculature of human patients who died of ischemic stroke due to systemic hypertension. Hematoxylin and eosin staining and immunohistochemistry showed the occurrence of brain vascular hyalinosis with infiltrated plasma proteins along with the narrowing of the vasa vasorum and oxidative stress. Transmission electron microscopy revealed endothelial cell bulge protrusion into the vasa vasorum lumen and the occurrence of endocytosis in the vasa vasorum endothelium. The treatment of cultured microvascular endothelial cells with adrenaline also promoted the formation of the bulge as well as endocytic vesicles. The siRNA knockdown of sortin nexin-9 (a mediator of clathrin-mediated endocytosis) inhibited adrenaline-induced endothelial cell bulge formation. Adrenaline promoted protein-protein interactions between sortin nexin-9 and neural Wiskott–Aldrich syndrome protein (a regulator of actin polymerization). Spontaneously hypertensive stroke-prone rats also exhibited lesions indicative of brain vascular hyalinosis, the endothelial cell protrusion into the lumen of the vasa vasorum, and endocytosis in vasa vasorum endothelial cells. We propose that endocytosis-dependent endothelial cell bulge protrusion narrows the vasa vasorum, resulting in ischemic oxidative damage to cerebral vessels, the formation of hyalinosis, the occurrence of ischemic stroke, and death in systemic hypertension patients.

scholarly journals Extracellular Vesicles Deliver Mitochondria and HSP27 Protein to Protect the Blood-Brain Barrier

2021 ◽  
Author(s):  
Kandarp Dave ◽  
Michael John Reynolds ◽  
Donna B Stolz ◽  
Riyan Babidhan ◽  
Duncan X Dobbins ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Ischemic Stroke ◽  
Endothelial Cell ◽  
Tight Junction ◽  
Human Brain ◽  
Blood Brain Barrier ◽  
Extracellular Vesicles ◽  
Brain Barrier ◽  
Brain Endothelial Cell ◽  
Brain Endothelial Cells

Ischemic stroke causes brain endothelial cell death and damages tight junction integrity of the blood-brain barrier (BBB). We engineered endothelial cell-derived extracellular vesicles (EVs) for the delivery of exogenous heat shock protein 27 (HSP27) and harnessed the innate EV mitochondrial load as a one, two-punch strategy to increase brain endothelial cell survival (via mitochondrial delivery) and preserve their tight junction integrity (via HSP27 delivery). We demonstrated that endothelial microvesicles but not exosomes transferred their mitochondrial load that subsequently underwent fusion with the mitochondrial network of the recipient primary human brain endothelial cells. This mitochondrial transfer increased the relative ATP levels and mitochondrial function in the recipient endothelial cells. EV-mediated HSP27 delivery to primary human brain endothelial cells decreased the paracellular permeability of small and large molecular mass fluorescent tracers in an in vitro model of ischemia/reperfusion injury. This one, two-punch approach to increase the metabolic function and structural integrity of brain endothelial cells is a promising strategy for BBB protection and prevention of long-term neurological dysfunction post-ischemic stroke. 

scholarly journals Is LRP2 Involved in Leptin Transport over the Blood-Brain Barrier and Development of Obesity?

2021 ◽  
Vol 22 (9) ◽  
pp. 4998
Author(s):  
Elvira S. Sandin ◽  
Julica Folberth ◽  
Helge Müller-Fielitz ◽  
Claus U. Pietrzik ◽  
Elisabeth Herold ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Blood Brain Barrier ◽  
Leptin Receptor ◽  
Brain Barrier ◽  
Blood Brain ◽  
In Vitro Bbb Model ◽  
Porcine Endothelial Cells ◽  
The Brain

The mechanisms underlying the transport of leptin into the brain are still largely unclear. While the leptin receptor has been implicated in the transport process, recent evidence has suggested an additional role of LRP2 (megalin). To evaluate the function of LRP2 for leptin transport across the blood-brain barrier (BBB), we developed a novel leptin-luciferase fusion protein (pLG), which stimulated leptin signaling and was transported in an in vitro BBB model based on porcine endothelial cells. The LRP inhibitor RAP did not affect leptin transport, arguing against a role of LRP2. In line with this, the selective deletion of LRP2 in brain endothelial cells and epithelial cells of the choroid plexus did not influence bodyweight, body composition, food intake, or energy expenditure of mice. These findings suggest that LRP2 at the BBB is not involved in the transport of leptin into the brain, nor in the development of obesity as has previously been described.

scholarly journals Mesenchymal Stem Cell Derived Extracellular Vesicles for Repairing the Neurovascular Unit after Ischemic Stroke

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 767
Author(s):  
Courtney Davis ◽  
Sean I. Savitz ◽  
Nikunj Satani
Keyword(s):  
Ischemic Stroke ◽  
Extracellular Vesicles ◽  
Neurovascular Unit ◽  
Culture Conditions ◽  
Therapeutic Effects ◽  
Stroke Treatment ◽  
Inflammatory Molecules ◽  
The Brain

Ischemic stroke is a debilitating disease and one of the leading causes of long-term disability. During the early phase after ischemic stroke, the blood-brain barrier (BBB) exhibits increased permeability and disruption, leading to an influx of immune cells and inflammatory molecules that exacerbate the damage to the brain tissue. Mesenchymal stem cells have been investigated as a promising therapy to improve the recovery after ischemic stroke. The therapeutic effects imparted by MSCs are mostly paracrine. Recently, the role of extracellular vesicles released by these MSCs have been studied as possible carriers of information to the brain. This review focuses on the potential of MSC derived EVs to repair the components of the neurovascular unit (NVU) controlling the BBB, in order to promote overall recovery from stroke. Here, we review the techniques for increasing the effectiveness of MSC-based therapeutics, such as improved homing capabilities, bioengineering protein expression, modified culture conditions, and customizing the contents of EVs. Combining multiple techniques targeting NVU repair may provide the basis for improved future stroke treatment paradigms.

scholarly journals Cardiac regeneration: epicardial mediated repair

2015 ◽  
Vol 282 (1821) ◽  
pp. 20152147 ◽  
Author(s):  
Teresa Kennedy-Lydon ◽  
Nadia Rosenthal
Keyword(s):  
Stem Cells ◽  
Endothelial Cells ◽  
Cardiac Fibroblasts ◽  
Cardiac Regeneration ◽  
Repair Process ◽  
Regenerative Process ◽  
Cardiac Stem Cell ◽  
Cardiomyocyte Proliferation ◽  
Lower Vertebrates

The hearts of lower vertebrates such as fish and salamanders display scarless regeneration following injury, although this feature is lost in adult mammals. The remarkable capacity of the neonatal mammalian heart to regenerate suggests that the underlying machinery required for the regenerative process is evolutionarily retained. Recent studies highlight the epicardial covering of the heart as an important source of the signalling factors required for the repair process. The developing epicardium is also a major source of cardiac fibroblasts, smooth muscle, endothelial cells and stem cells. Here, we examine animal models that are capable of scarless regeneration, the role of the epicardium as a source of cells, signalling mechanisms implicated in the regenerative process and how these mechanisms influence cardiomyocyte proliferation. We also discuss recent advances in cardiac stem cell research and potential therapeutic targets arising from these studies.

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