| 1. |
- Alvarez, Alberto, et al.
(författare)
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Cdc42 is required in mural cells for proper patterning of the retinal vasculature
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Mural cells constitute the outer lining of blood vessels. They are known as pericytes in the capillary network and referred to as smooth muscle cells (SMC) around arteries and veins. Despite their ubiquity, their contribution to vascular morphogenesis remains obscure. In this work, we investigated the role of Cdc42 in mural cells in vivo, using the developing mouse retina as a model. We find that, during postnatal development, Cdc42 is required in both, pericytes and smooth muscle cells to maintain proper cell morphology, coverage and distribution. During retinal angiogenesis, Cdc42-depleted pericytes lag behind the sprouting front, at least in part due to decreased proliferation. Consequently, capillaries at the sprouting front remain pericyte deprived and are prone to increased vascular leakage. In addition, arteries and arterioles deviate from their normal growth directions and trajectory. While in the adult retina, mural cell coverage normalizes and pericytes adopt a normal morphology, smooth muscle cell morphologies remain abnormal and arteriolar branching angles are markedly reduced. Our findings demonstrate that Cdc42 is required for mural cell proliferation, morphology and distribution and suggest that mural cells are essential for normal vascular morphogenesis of the developing retinal vasculature.
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| 2. |
- Alvarez, Alberto, et al.
(författare)
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Tamoxifen-independent recombination of reporter genes limits lineage tracing and mosaic analysis using CreER(T2) lines
- 2020
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Ingår i: Transgenic research. - : Springer Nature. - 0962-8819 .- 1573-9368. ; 29:1, s. 53-68
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Tidskriftsartikel (refereegranskat)abstract
- The CreER(T2)/loxP system is widely used to induce conditional gene deletion in mice. One of the main advantages of the system is that Cre-mediated recombination can be controlled in time through Tamoxifen administration. This has allowed researchers to study the function of embryonic lethal genes at later developmental timepoints. In addition, CreER(T2) mouse lines are commonly used in combination with reporter genes for lineage tracing and mosaic analysis. In order for these experiments to be reliable, it is crucial that the cell labeling approach only marks the desired cell population and their progeny, as unfaithful expression of reporter genes in other cell types or even unintended labeling of the correct cell population at an undesired time point could lead to wrong conclusions. Here we report that all CreER(T2) mouse lines that we have studied exhibit a certain degree of Tamoxifen-independent, basal, Cre activity. Using Ai14 and Ai3, two commonly used fluorescent reporter genes, we show that those basal Cre activity levels are sufficient to label a significant amount of cells in a variety of tissues during embryogenesis, postnatal development and adulthood. This unintended labelling of cells imposes a serious problem for lineage tracing and mosaic analysis experiments. Importantly, however, we find that reporter constructs differ greatly in their susceptibility to basal CreER(T2) activity. While Ai14 and Ai3 easily recombine under basal CreER(T2) activity levels, mTmG and R26R-EYFP rarely become activated under these conditions and are therefore better suited for cell tracking experiments.
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| 3. |
- Álvarez-Aznar, Alberto
(författare)
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Cdc42, orchestrator of vascular morphogenesis in the retina
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cdc42 is a small GTPase that controls many cellular functions related to cytoskeletal dynamics, such as migration, polarity, and proliferation. Despite what we know of Cdc42 in other cell types, not much research has been done on the vasculature. This thesis describes the consequences of Cdc42 deletion in two vascular cell types—endothelial and mural cells—during developmental angiogenesis.In paper I, we demonstrate through a combination of in vitro, in silico, and in vivo assays, that Cdc42-deficient endothelial cells migrate less and fail to distribute normally in areas of naturally occurring high proliferation during angiogenesis, causing vascular malformations with enlarged lumens. In addition, these cells present impaired filopodia formation, a disadvantage for the tip cell position, disturbed axial polarity and altered junctions.With an in vivo approach, in paper III we demonstrate that the deletion of Cdc42 in mural cells has consequences on the morphogenesis of the retinal vasculature. Cdc42-deficient mural cells proliferate less and cannot keep up with the nascent angiogenic vasculature, which results in a complete pericyte loss at the sprouting front. Furthermore, we describe that mural cells contribute to the remodeling of the vasculature, also after the initial phases of angiogenesis.The CreERT2 system is frequently used for conditional gene deletion and lineage tracing. Tamoxifen administration allows spatiotemporally controlled recombination of fluorescent reporters, and tracing of the labeled cells. However, in the course of our studies, we observed tamoxifen-independent recombination. In paper II, we describe this phenomenon in detail, using different combinations of CreERT2 and fluorescent reporter lines. We conclude that tamoxifen-independent recombination is a widespread occurrence, and that fluorescent reporter lines present varying levels of susceptibility to it.In summary, the work presented here sheds new light on the role of Cdc42 in the vasculature. Additionally, this thesis describes in detail an important feature of CreERT2 and reporter lines that should be taken into account when performing lineage-tracing experiments.
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| 4. |
- Betsholtz, Christer, et al.
(författare)
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Cellular Origin of Sporadic CCMs
- 2022
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Ingår i: New England Journal of Medicine. - : Massachusetts Medical Society. - 0028-4793 .- 1533-4406. ; 386:13, s. 1291-1291
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 5. |
- 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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| 6. |
- Collu, Giovanna M., et al.
(författare)
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Prickle is phosphorylated by Nemo and targeted for degradation to maintain Prickle/Spiny-legs isoform balance during planar cell polarity establishment
- 2018
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Ingår i: PLOS Genetics. - : PUBLIC LIBRARY SCIENCE. - 1553-7390 .- 1553-7404. ; 14:5
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Tidskriftsartikel (refereegranskat)abstract
- Planar cell polarity (PCP) instructs tissue patterning in a wide range of organisms from fruit flies to humans. PCP signaling coordinates cell behavior across tissues and is integrated by cells to couple cell fate identity with position in a developing tissue. In the fly eye, PCP signaling is required for the specification of R3 and R4 photoreceptors based upon their positioning relative to the dorso-ventral axis. The 'core' PCP pathway involves the asymmetric localization of two distinct membrane-bound complexes, one containing Frizzled (Fz, required in R3) and the other Van Gogh (Vang, required in R4). Inhibitory interactions between the cytosolic components of each complex reinforce asymmetric localization. Prickle (Pk) and Spiny-legs (Pk-Sple) are two antagonistic isoforms of the prickle (pk) gene and are cytoplasmic components of the Vang complex. The balance between their levels is critical for tissue patterning, with Pk-Sple being the major functional isoform in the eye. Here we uncover a post-translational role for Nemo kinase in limiting the amount of the minor isoform Pk. We identified Pk as a Nemo substrate in a genome-wide in vitro band-shift screen. In vivo, nemo genetically interacts with pk(pk) but not pk(sple) and enhances PCP defects in the eye and leg. Nemo phosphorylation limits Pk levels and is required specifically in the R4 photoreceptor like the major isoform, Pk-Sple. Genetic interaction and biochemical data suggest that Nemo phosphorylation of Pk leads to its proteasomal degradation via the Cullin1/SkpA/Slmb complex. dTAK and Homeodomain interacting protein kinase (Hipk) may also act together with Nemo to target Pk for degradation, consistent with similar observations in mammalian studies. Our results therefore demonstrate a mechanism to maintain low levels of the minor Pk isoform, allowing PCP complexes to form correctly and specify cell fate.
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| 7. |
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| 8. |
- Fernandez-Chacon, Macarena, et al.
(författare)
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Incongruence between transcriptional and vascular pathophysiological cell states
- 2023
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Ingår i: NATURE CARDIOVASCULAR RESEARCH. - : SPRINGERNATURE. - 2731-0590. ; 2:6, s. 530-549
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Tidskriftsartikel (refereegranskat)abstract
- The Notch pathway is a major regulator of endothelial transcriptional specification. Targeting the Notch receptors or Delta-like ligand 4 (Dll4) dysregulates angiogenesis. Here, by analyzing single and compound genetic mutants for all Notch signaling members, we find significant differences in the way ligands and receptors regulate liver vascular homeostasis. Loss of Notch receptors caused endothelial hypermitogenic cell-cycle arrest and senescence. Conversely, Dll4 loss triggered a strong Myc-driven transcriptional switch inducing endothelial proliferation and the tip-cell state. Myc loss suppressed the induction of angiogenesis in the absence of Dll4, without preventing the vascular enlargement and organ pathology. Similarly, inhibition of other pro-angiogenic pathways, including MAPK/ERK and mTOR, had no effect on the vascular expansion induced by Dll4 loss; however, anti-VEGFA treatment prevented it without fully suppressing the transcriptional and metabolic programs. This study shows incongruence between single-cell transcriptional states, vascular phenotypes and related pathophysiology. Our findings also suggest that the vascular structure abnormalization, rather than neoplasms, causes the reported anti-Dll4 antibody toxicity. Fernandez-Chacon et al. use imaging and scRNA-seq after targeting multiple Notch genes and angiogenic signaling pathways to find that the function of these pathways in vascular pathophysiology cannot be predicted by assessing transcriptional states.
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| 9. |
- Gängel, Konstantin, et al.
(författare)
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Endocytosis regulates VEGF signalling during angiogenesis
- 2013
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Ingår i: Nature Cell Biology. - : Springer Science and Business Media LLC. - 1465-7392 .- 1476-4679. ; 15:3, s. 233-235
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Endocytosis has proved to be a versatile mechanism regulating diverse cellular processes, ranging from nutrient uptake to intracellular signal transduction. New work reinforces the importance of endocytosis for VEGF receptor signalling and angiogenesis in the developing eye, and describes a mechanism for its differential regulation in angiogenic versus quiescent endothelial cells.
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| 10. |
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