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| 2. |
- Castro, Marco, et al.
(författare)
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CDC42 deletion elicits cerebral vascular malformations via increased MEKK3-dependent KLF4 expression
- 2019
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Ingår i: Circulation Research. - 0009-7330 .- 1524-4571. ; 124:8, s. 1240-1252
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Tidskriftsartikel (refereegranskat)abstract
- Rationale: Aberrant formation of blood vessels precedes a broad spectrum of vascular complications; however, the cellular and molecular events governing vascular malformations are not yet fully understood. Objective: Here, we investigated the role of CDC42 (cell division cycle 42) during vascular morphogenesis and its relative importance for the development of cerebrovascular malformations. Methods and Results: To avoid secondary systemic effects often associated with embryonic gene deletion, we generated an endothelial-specific and inducible knockout approach to study postnatal vascularization of the mouse brain. Postnatal endothelial-specific deletion of Cdc42 elicits cerebrovascular malformations reminiscent of cerebral cavernous malformations (CCMs). At the cellular level, loss of CDC42 function in brain endothelial cells (ECs) impairs their sprouting, branching morphogenesis, axial polarity, and normal dispersion within the brain tissue. Disruption of CDC42 does not alter EC proliferation, but malformations occur where EC proliferation is the most pronounced during brain development-the postnatal cerebellum-indicating that a high, naturally occurring EC proliferation provides a permissive state for the appearance of these malformations. Mechanistically, CDC42 depletion in ECs elicited increased MEKK3 (mitogen-activated protein kinase kinase kinase 3)-MEK5 (mitogen-activated protein kinase kinase 5)-ERK5 (extracellular signal-regulated kinase 5) signaling and consequent detrimental overexpression of KLF (Kruppel-like factor) 2 and KLF4, recapitulating the hallmark mechanism for CCM pathogenesis. Through genetic approaches, we demonstrate that the coinactivation of Klf4 reduces the severity of vascular malformations in Cdc42 mutant mice. Moreover, we show that CDC42 interacts with CCMs and that CCM3 promotes CDC42 activity in ECs. Conclusions: We show that endothelial-specific deletion of Cdc42 elicits CCM-like cerebrovascular malformations and that CDC42 is engaged in the CCM signaling network to restrain the MEKK3-MEK5-ERK5-KLF2/4 pathway.
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| 3. |
- Castro, Marco
(författare)
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Cellular and molecular roles for CDC42 in angiogenesis
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Angiogenesis is the physiological process by which new blood vessels grow and critically depends on the interplay between the major vascular units: endothelial cells, pericytes and smooth muscle cells. Dysfunction and mispatterning of blood vessels are associated with the progression of many vascular complications, and therefore, understanding the causes of vascular dysmorphia is a central question in vascular biology. CDC42 is a small GTPase known to regulate a diverse array of cellular functions in endothelial cells, however, its contribution to vascular development in vivo remains incompletely understood. The overall aim of this thesis work is to investigate the role of CDC42 during angiogenesis in the central nervous system, using an inducible endothelial-specific Cdc42 knockout model.In Paper I, I investigate which CDC42-dependent functions operational in vivo are of relevance for angiogenic sprouting, and how they contribute to blood vessel morphogenesis. Analysis of distinct cellular behaviours shows that CDC42 is critically required for proper EC dispersion in the vasculature and that it regulates sprouting angiogenesis and endothelial axial polarity.In Paper II, I explore the in vivo consequences of Cdc42 deletion for vascular morphogenesis, leading to the appearance of capillary-venous malformations in the brain, resembling the human disease of cerebral cavernous malformations. I aimed to understand how this type of vascular malformations arise and was been able to identify the MEKK3-ERK5-KLF2/4 molecular signalling pathway and other cellular events as the trigger factors that may be responsible for these malformations.Paper III redirects focus to the physiological roles of another protein, GPR116, in modulating blood-brain barrier permeability and pathologic angiogenesis in the central nervous system.In summary, these findings reveal crucial roles of endothelial CDC42 during angiogenesis and further uncover its potential relevance in the molecular pathogenesis of cerebrovascular malformations.
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| 5. |
- He, Liqun, et al.
(författare)
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Single-cell RNA sequencing of mouse brain and lung vascular and vessel-associated cell types
- 2018
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Ingår i: Scientific Data. - : NATURE PUBLISHING GROUP. - 2052-4463. ; 5
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Tidskriftsartikel (refereegranskat)abstract
- Vascular diseases are major causes of death, yet our understanding of the cellular constituents of blood vessels, including how differences in their gene expression profiles create diversity in vascular structure and function, is limited. In this paper, we describe a single-cell RNA sequencing (scRNA-seq) dataset that defines vascular and vessel-associated cell types and subtypes in mouse brain and lung. The dataset contains 3,436 single cell transcriptomes from mouse brain, which formed 15 distinct clusters corresponding to cell (sub) types, and another 1,504 single cell transcriptomes from mouse lung, which formed 17 cell clusters. In order to allow user-friendly access to our data, we constructed a searchable database (http://betsholtzlab.org/VascularSingleCells/database.html). Our dataset constitutes a comprehensive molecular atlas of vascular and vessel-associated cell types in the mouse brain and lung, and as such provides a strong foundation for future studies of vascular development and diseases.
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| 6. |
- Herrera-Hidalgo, Carmen, et al.
(författare)
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Macrophages contribute to vessel normalization during healing of ischemic injury
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Physiological regression of superfluous micro-vessels, vascular pruning, is observed during formation of functional vascular networks in fetal development. The current study investigates the vascular dynamics during healing of injured adult tissues in the mouse model of hind limb ischemia. We found that prompt angiogenesis that occurs in M. gastrocnemius to compensate for loss of tissue perfusion, results in blood vessel densities that are higher than those found in healthy muscles. Vessel density peaked at day 14 post ischemia to thereafter recede to normal levels by day 21, indicating that vessel pruning occurs during healing to ensure establishment of an optimal vascular tree. Macrophages are professional phagocytes and we found them present in high numbers at sites of ischemic injury, where they were positioned in close contact with the vascular network and displayed phagocytic activity at the time of vessel pruning. Interestingly, macrophage depletion between day 14 and 21 post-ischemia resulted in reduced vessel regression. Indeed, by using a reporter mouse for endothelial cells, we found that macrophages engulf endothelial cells at the ischemic site at this time point. Taken together, our results indicate a role for macrophages in vessel normalization by pruning superfluous vasculatur segments that form initially during healing of ischemic injuries.
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| 7. |
- Honkura, Naoki, et al.
(författare)
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Intravital imaging-based analysis tools for vessel identification and assessment of concurrent dynamic vascular events
- 2018
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Ingår i: Nature communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 9:1, s. 2746-
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Tidskriftsartikel (refereegranskat)abstract
- The vasculature undergoes changes in diameter, permeability and blood flow in response to specific stimuli. The dynamics and interdependence of these responses in different vessels are largely unknown. Here we report a non-invasive technique to study dynamic events in different vessel categories by multi-photon microscopy and an image analysis tool, RVDM (relative velocity, direction, and morphology) allowing the identification of vessel categories by their red blood cell (RBC) parameters. Moreover, Claudin5 promoter-driven green fluorescent protein (GFP) expression is used to distinguish capillary subtypes. Intradermal injection of vascular endothelial growth factor A (VEGFA) is shown to induce leakage of circulating dextran, with vessel-type-dependent kinetics, from capillaries and venules devoid of GFP expression. VEGFA-induced leakage in capillaries coincides with vessel dilation and reduced flow velocity. Thus, intravital imaging of non-invasive stimulation combined with RVDM analysis allows for recording and quantification of very rapid events in the vasculature.
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| 8. |
- Huang, Hua, 1986-, et al.
(författare)
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ELTD1-deletion reduces vascular abnormality and improves T-cell recruitment after PD-1 blockade in glioma.
- 2021
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Ingår i: Neuro-Oncology. - : Oxford University Press. - 1522-8517 .- 1523-5866. ; 24:3, s. 398-411
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Tumor vessels in glioma are molecularly and functionally abnormal, contributing to treatment resistance. Proteins differentially expressed in glioma vessels can change vessel phenotype and be targeted for therapy. ELTD1 (Adgrl4) is an orphan member of the adhesion G-protein-coupled receptor family upregulated in glioma vessels, and has been suggested as a potential therapeutic target. However, the role of ELTD1 in regulating vessel function in glioblastoma is poorly understood.METHODS: ELTD1 expression in human gliomas and its association with patient survival was determined using tissue microarrays and public databases. The role of ELTD1 in regulating tumor vessel phenotype was analyzed using orthotopic glioma models and ELTD1 -/- mice. Endothelial cells isolated from murine gliomas were transcriptionally profiled to determine differentially expressed genes and pathways. The consequence of ELTD1-deletion on glioma immunity was determined by treating tumor bearing mice with PD-1-blocking antibodies.RESULTS: ELTD1 levels were upregulated in human glioma vessels, increased with tumor malignancy, and were associated with poor patient survival. Progression of orthotopic gliomas was not affected by ELTD1-deletion, however, tumor vascular function was improved in ELTD1 -/- mice. Bioinformatic analysis of differentially expressed genes indicated increased inflammatory response and decreased proliferation in tumor endothelium in ELTD1 -/- mice. Consistent with an enhanced inflammatory response, ELTD1-deletion improved T-cell infiltration in GL261-bearing mice after PD-1 checkpoint blockade.CONCLUSION: Our data demonstrate that ELTD1 participates in inducing vascular dysfunction in glioma, and suggests that targeting of ELTD1 may normalize the vessels and improve the response to immunotherapy.
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| 10. |
- Laviña, Bàrbara
(författare)
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Brain Vascular Imaging Techniques
- 2017
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Ingår i: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 18:1
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Forskningsöversikt (refereegranskat)abstract
- Recent major improvements in a number of imaging techniques now allow for the study of the brain in ways that could not be considered previously. Researchers today have well-developed tools to specifically examine the dynamic nature of the blood vessels in the brain during development and adulthood; as well as to observe the vascular responses in disease situations in vivo. This review offers a concise summary and brief historical reference of different imaging techniques and how these tools can be applied to study the brain vasculature and the blood-brain barrier integrity in both healthy and disease states. Moreover, it offers an overview on available transgenic animal models to study vascular biology and a description of useful online brain atlases.
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