| 1. |
- Alvarez, Alberto, et al.
(författare)
-
Tamoxifen-independent recombination of reporter genes limits lineage tracing and mosaic analysis using CreER(T2) lines
- 2020
-
Ingår i: Transgenic research. - : Springer Nature. - 0962-8819 .- 1573-9368. ; 29:1, s. 53-68
-
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.
|
|
| 2. |
- Álvarez-Aznar, Alberto
(författare)
-
Cdc42, orchestrator of vascular morphogenesis in the retina
- 2020
-
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.
|
|
| 3. |
- Arasa, Jorge, et al.
(författare)
-
Upregulation of VCAM-1 in lymphatic collectors supports dendritic cell entry and rapid migration to lymph nodes in inflammation
- 2021
-
Ingår i: Journal of Experimental Medicine. - : Rockefeller University Press. - 0022-1007 .- 1540-9538. ; 218:7
-
Tidskriftsartikel (refereegranskat)abstract
- Dendritic cell (DC) migration to draining lymph nodes (dLNs) is a slow process that is believed to begin with DCs approaching and entering into afferent lymphatic capillaries. From capillaries, DCs slowly crawl into lymphatic collectors, where lymph flow induced by collector contraction supports DC detachment and thereafter rapid, passive transport to dLNs. Performing a transcriptomics analysis of dermal endothelial cells, we found that inflammation induces the degradation of the basement membrane (BM) surrounding lymphatic collectors and preferential up-regulation of the DC trafficking molecule VCAM-1 in collectors. In crawl-in experiments performed in ear skin explants, DCs entered collectors in a CCR7- and beta 1 integrin-dependent manner. In vivo, loss of beta 1-integrins in DCs or of VCAM-1 in lymphatic collectors had the greatest impact on DC migration to dLNs at early time points when migration kinetics favor the accumulation of rapidly migrating collector DCs rather than slower capillary DCs. Taken together, our findings identify collector entry as a critical mechanism enabling rapid DC migration to dLNs in inflammation.
|
|
| 4. |
- Betsholtz, Christer, et al.
(författare)
-
Cellular Origin of Sporadic CCMs
- 2022
-
Ingår i: New England Journal of Medicine. - : Massachusetts Medical Society. - 0028-4793 .- 1533-4406. ; 386:13, s. 1291-1291
-
Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
|
|
| 5. |
- Chen, Di, et al.
(författare)
-
Angiogenesis depends upon EPHB4-mediated export of collagen IV from vascular endothelial cells
- 2022
-
Ingår i: JCI Insight. - : American Society for Clinical Investigation. - 2379-3708. ; 7:4
-
Tidskriftsartikel (refereegranskat)abstract
- Capillary malformation-arteriovenous malformation (CM-AVM) is a blood vascular anomaly caused by inherited loss-of-function mutations in RASA1 or EPHB4 genes, which encode p120 Ras GTPase-activating protein (p120 RasGAP/RASA1) and Ephrin receptor B4 (EPHB4). However, whether RASA1 and EPHB4 function in the same molecular signaling pathway to regulate the blood vasculature is uncertain. Here, we show that induced endothelial cell-specific (EC-specific) disruption of Ephb4 in mice resulted in accumulation of collagen IV in the EC ER, leading to EC apoptotic death and defective developmental, neonatal, and pathological angiogenesis, as reported previously in induced EC-specific RASA1-deficient mice. Moreover, defects in angiogenic responses in EPHB4-deficient mice could be rescued by drugs that inhibit signaling through the Ras pathway and drugs that promote collagen IV export from the ER. However, EPHB4-mutant mice that expressed a form of EPHB4 that is unable to physically engage RASA1 but retains protein tyrosine kinase activity showed normal angiogenic responses. These findings provide strong evidence that RASA1 and EPHB4 function in the same signaling pathway to protect against the development of CM-AVM independent of physical interaction and have important implications for possible means of treatment of this disease.
|
|
| 6. |
- Daubel, Nina
(författare)
-
Lymphatic Vascular Morphogenesis : From Progenitors to Functional Vessels
- 2022
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The lymphatic vasculature is an important part of the circulatory system and crucial for normal functioning and maintenance of tissues. Yet, our understanding of the processes underlying lymphatic development and homeostasis are surprisingly limited. Recent studies have uncovered a heterogeneous origin of lymphatic endothelium within different organs as well as different mechanisms of vessel formation. The mesentery is a fold of peritoneum that attaches the intestines to the abdominal wall and harbours nerves, blood and lymphatic vessels which supply the intestines. Here, the lymphatic vasculature forms through a process termed lymphvasculogenesis, during which non-venous derived lymphatic endothelial cell (LEC) progenitors assemble into vessels. Parallel to this process, the mesenteric blood vasculature undergoes extensive remodelling. In paper II we show that this is accompanied by a transient extravasation of red blood cells (RBCs). Engulfment of RBCs by developing lymphatic vessels indicate a novel role of lymphatics in clearance of extravasated RBCs. In paper III we further analyse early LEC progenitors in the mesentery and show that they exhibit unique characteristics including membrane blebbing that may facilitate LEC migration during lymphvasculogenic vessel formation. The primitive lymphatic plexus further develops into mature vessels with blind ended, highly specialized segments termed lymphatic capillaries. Individual capillary LECs possess a characteristic oak leaf like shape and discontinuous button like junctions. In paper IV we propose a new model of cell shape regulation in lymphatic capillaries that is based on the interplay of the cytoskeleton and a unique organization of cell-cell junctions. We further report that acquisition of oak leaf shape precedes junctional specification, and is not a mere result of button junction formation in dermal lymphatics. CreERT2 mouse lines are used across many fields of biological research, including the here presented studies, because they allow for targeted gene deletion upon inducible genetic recombination. In paper I we report that, unexpectedly, several commonly used CreERT2 mouse lines exhibit a weak baseline Cre activity leading to induction-independent recombination. This has important implications for the interpretation of results from Cre/loxP experiments, especially when performing lineage tracing.Focusing on different aspects of lymphatic vascular biology, this thesis work reveals yet undescribed mechanisms by which LECs form new vessels, contribute to tissue integrity during vascular remodelling and maintain mature lymphatic vessel integrity through a unique interplay of cell shape and junctional organization.
|
|
| 7. |
- Frye, Maike, et al.
(författare)
-
EphrinB2-EphB4 signalling provides Rho-mediated homeostatic control of lymphatic endothelial cell junction integrity
- 2020
-
Ingår i: eLIFE. - : eLife Sciences Publications, Ltd. - 2050-084X. ; 9
-
Tidskriftsartikel (refereegranskat)abstract
- Endothelial integrity is vital for homeostasis and adjusted to tissue demands. Although fluid uptake by lymphatic capillaries is a critical attribute of the lymphatic vasculature, the barrier function of collecting lymphatic vessels is also important by ensuring efficient fluid drainage as well as lymph node delivery of antigens and immune cells. Here, we identified the transmembrane ligand EphrinB2 and its receptor EphB4 as critical homeostatic regulators of collecting lymphatic vessel integrity. Conditional gene deletion in mice revealed that EphrinB2/EphB4 signalling is dispensable for blood endothelial barrier function, but required for stabilization of lymphatic endothelial cell (LEC) junctions in different organs of juvenile and adult mice. Studies in primary human LECs further showed that basal EphrinB2/EphB4 signalling controls junctional localisation of the tight junction protein CLDN5 and junction stability via Rac1/Rho-mediated regulation of cytoskeletal contractility. EphrinB2/EphB4 signalling therefore provides a potential therapeutic target to selectively modulate lymphatic vessel permeability and function.
|
|
| 8. |
- Hernández Vásquez, Magda, et al.
(författare)
-
Transcription factor FOXP2 is a flow-induced regulator of collecting lymphatic vessels
- 2021
-
Ingår i: EMBO Journal. - : EMBO Press. - 0261-4189 .- 1460-2075. ; 40:12
-
Tidskriftsartikel (refereegranskat)abstract
- The lymphatic system is composed of a hierarchical network of fluid absorbing lymphatic capillaries and transporting collecting vessels. Despite distinct functions and morphologies, molecular mechanisms that regulate the identity of the different vessel types are poorly understood. Through transcriptional analysis of murine dermal lymphatic endothelial cells (LECs), we identified Foxp2, a member of the FOXP family of transcription factors implicated in speech development, as a collecting vessel signature gene. FOXP2 expression was induced after initiation of lymph flow in vivo and upon shear stress on primary LECs in vitro. Loss of FOXC2, the major flow-responsive transcriptional regulator of lymphatic valve formation, abolished FOXP2 induction in vitro and in vivo. Genetic deletion of Foxp2 in mice using the endothelial-specific Tie2-Cre or the tamoxifen-inducible LEC-specific Prox1-CreERT2 line resulted in enlarged collecting vessels and defective valves characterized by loss of NFATc1 activity. Our results identify FOXP2 as a new flow-induced transcriptional regulator of collecting lymphatic vessel morphogenesis and highlight the existence of unique transcription factor codes in the establishment of vessel-type-specific endothelial cell identities.
|
|
| 9. |
- Korhonen, Emilia A., et al.
(författare)
-
Lymphangiogenesis requires Ang2/Tie/PI3K signaling for VEGFR3 cell-surface expression
- 2022
-
Ingår i: Journal of Clinical Investigation. - : American Society for Clinical Investigation. - 0021-9738 .- 1558-8238. ; 132:15
-
Tidskriftsartikel (refereegranskat)abstract
- Vascular endothelial growth factor C (VEGF-C) induces lymphangiogenesis via VEGF receptor 3 (VEGFR3), which is encoded by the most frequently mutated gene in human primary lymphedema. Angiopoietins (Angs) and their Tie receptors regulate lymphatic vessel development, and mutations of the ANGPT2 gene were recently found in human primary lymphedema. However, the mechanistic basis of Ang2 activity in lymphangiogenesis is not fully understood. Here, we used gene deletion, blocking Abs, transgene induction, and gene transfer to study how Ang2, its Tie2 receptor, and Tie1 regulate lymphatic vessels. We discovered that VEGF-C???induced Ang2 secretion from lymphatic endothelial cells (LECs) was involved in full Akt activation downstream of phosphoinositide 3 kinase (PI3K). Neonatal deletion of genes encoding the Tie receptors or Ang2 in LECs, or administration of an Ang2-blocking Ab decreased VEGFR3 presentation on LECs and inhibited lymphangiogenesis. A similar effect was observed in LECs upon deletion of the PI3K catalytic p110?? subunit or with small -molecule inhibition of a constitutively active PI3K located downstream of Ang2. Deletion of Tie receptors or blockade of Ang2 decreased VEGF-C???induced lymphangiogenesis also in adult mice. Our results reveal an important crosstalk between the VEGF-C and Ang signaling pathways and suggest new avenues for therapeutic manipulation of lymphangiogenesis by targeting Ang2/Tie/PI3K signaling.
|
|
| 10. |
- Lyons, Oliver, et al.
(författare)
-
Mutations in EPHB4 cause human venous valve aplasia
- 2021
-
Ingår i: JCI Insight. - : American Society For Clinical Investigation. - 2379-3708. ; 6:18
-
Tidskriftsartikel (refereegranskat)abstract
- Venous valve (VV) failure causes chronic venous insufficiency, but the molecular regulation of valve development is poorly understood. A primary lymphatic anomaly, caused by mutations in the receptor tyrosine kinase EPHB4, was recently described, with these patients also presenting with venous insufficiency. Whether the venous anomalies are the result of an effect on VVs is not known. VV formation requires complex "organization" of valve-forming endothelial cells, including their reorientation perpendicular to the direction of blood flow. Using quantitative ultrasound, we identified substantial VV aplasia and deep venous reflux in patients with mutations in EPHB4. We used a GFP reporter in mice to study expression of its ligand, ephrinB2, and analyzed developmental phenotypes after conditional deletion of floxed Ephb4 and Efnb2 alleles. EphB4 and ephrinB2 expression patterns were dynamically regulated around organizing valve-forming cells. Efnb2 deletion disrupted the normal endothelial expression patterns of the gap junction proteins connexin37 and connexin43 (both required for normal valve development) around reorientating valve-forming cells and produced deficient valve-forming cell elongation, reorientation, polarity, and proliferation. Ephb4 was also required for valve-forming cell organization and subsequent growth of the valve leaflets. These results uncover a potentially novel cause of primary human VV aplasia.
|
|
| 11. |
- Martinez-Corral, Ines, et al.
(författare)
-
Blockade of VEGF-C signaling inhibits lymphatic malformations driven by oncogenic PIK3CA mutation
- 2020
-
Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 11:1
-
Tidskriftsartikel (refereegranskat)abstract
- Lymphatic malformations (LMs) are debilitating vascular anomalies presenting with large cysts (macrocystic) or lesions that infiltrate tissues (microcystic). Cellular mechanisms underlying LM pathology are poorly understood. Here we show that the somatic PIK3CA(H1047R) mutation, resulting in constitutive activation of the p110 alpha PI3K, underlies both macrocystic and microcystic LMs in human. Using a mouse model of PIK3CA(H1047R)-driven LM, we demonstrate that both types of malformations arise due to lymphatic endothelial cell (LEC)-autonomous defects, with the developmental timing of p110 alpha activation determining the LM subtype. In the postnatal vasculature, PIK3CA(H1047R) promotes LEC migration and lymphatic hypersprouting, leading to microcystic LMs that grow progressively in a vascular endothelial growth factor C (VEGF-C)-dependent manner. Combined inhibition of VEGF-C and the PI3K downstream target mTOR using Rapamycin, but neither treatment alone, promotes regression of lesions. The best therapeutic outcome for LM is thus achieved by co-inhibition of the upstream VEGF-C/VEGFR3 and the downstream PI3K/mTOR pathways. Lymphatic malformation (LM) is a debilitating often incurable vascular disease. Using a mouse model of LM driven by a disease-causative PIK3CA mutation, the authors show that vascular growth is dependent on the upstream lymphangiogenic VEGF-C signalling, permitting effective therapeutic intervention.
|
|
| 12. |
- Muhl, Lars, et al.
(författare)
-
The SARS-CoV-2 receptor ACE2 is expressed in mouse pericytes but not endothelial cells : Implications for COVID-19 vascular research
- 2022
-
Ingår i: Stem Cell Reports. - : Elsevier. - 2213-6711. ; 17:5, s. 1089-1104
-
Tidskriftsartikel (refereegranskat)abstract
- Humanized mouse models and mouse-adapted SARS-CoV-2 virus are increasingly used to study COVID-19 pathogenesis, so it is impor-tant to learn where the SARS-CoV-2 receptor ACE2 is expressed. Here we mapped ACE2 expression during mouse postnatal development and in adulthood. Pericytes in the CNS, heart, and pancreas express ACE2 strongly, as do perineurial and adrenal fibroblasts, whereas endothelial cells do not at any location analyzed. In a number of other organs, pericytes do not express ACE2, including in the lung where ACE2 instead is expressed in bronchial epithelium and alveolar type II cells. The onset of ACE2 expression is organ specific: in bronchial epithelium already at birth, in brain pericytes before, andin heart pericytes after postnatal day 10.5. Establishing the vascular localization of ACE2 expression is central to correctly interpret data from modeling COVID-19 in the mouse and may shed light on the cause of vascular COVID-19 complications.
|
|
| 13. |
|
|
| 14. |
- Mäkinen, Taija, et al.
(författare)
-
Lymphatic Malformations : Genetics, Mechanisms and Therapeutic Strategies
- 2021
-
Ingår i: Circulation Research. - : Lippincott Williams & Wilkins. - 0009-7330 .- 1524-4571. ; 129:1, s. 136-154
-
Forskningsöversikt (refereegranskat)abstract
- Lymphatic vessels maintain tissue fluid homeostasis by returning to blood circulation interstitial fluid that has extravasated from the blood capillaries. They provide a trafficking route for cells of the immune system, thus critically contributing to immune surveillance. Developmental or functional defects in the lymphatic vessels, their obstruction or damage, lead to accumulation of fluid in tissues, resulting in lymphedema. Here we discuss developmental lymphatic anomalies called lymphatic malformations and complex lymphatic anomalies that manifest as localized or multifocal lesions of the lymphatic vasculature, respectively. They are rare diseases that are caused mostly by somatic mutations and can present with variable symptoms based upon the size and location of the lesions composed of fluid-filled cisterns or channels. Substantial progress has been made recently in understanding the molecular basis of their pathogenesis through the identification of their genetic causes, combined with the elucidation of the underlying mechanisms in animal disease models and patient-derived lymphatic endothelial cells. Most of the solitary somatic mutations that cause lymphatic malformations and complex lymphatic anomalies occur in genes that encode components of oncogenic growth factor signal transduction pathways. This has led to successful repurposing of some targeted cancer therapeutics to the treatment of lymphatic malformations and complex lymphatic anomalies. Apart from the mutations that act as lymphatic endothelial cell-autonomous drivers of these anomalies, current evidence points to superimposed paracrine mechanisms that critically contribute to disease pathogenesis and thus provide additional targets for therapeutic intervention. Here, we review these advances and discuss new treatment strategies that are based on the recently identified molecular pathways.
|
|
| 15. |
- Ortsäter, Henrik, et al.
(författare)
-
An inducible Cldn11-CreERT2 mouse line for selective targeting of lymphatic valves
- 2021
-
Ingår i: Genesis. - : Wiley. - 1526-954X .- 1526-968X. ; 59:7-8
-
Tidskriftsartikel (refereegranskat)abstract
- Luminal valves of collecting lymphatic vessels are critical for maintaining unidirectional flow of lymph and their dysfunction underlies several forms of primary lymphedema. Here, we report on the generation of a transgenic mouse expressing the tamoxifen inducible CreERT2 under the control of Cldn11 promoter that allows, for the first time, selective and temporally controlled targeting of lymphatic valve endothelial cells. We show that within the vasculature CLDN11 is specifically expressed in lymphatic valves but is not required for their development as mice with a global loss of Cldn11 display normal valves in the mesentery. Tamoxifen treated Cldn11-CreERT2 mice also carrying a fluorescent Cre-reporter displayed reporter protein expression selectively in lymphatic valves and, to a lower degree, in venous valves. Analysis of developing vasculature further showed that Cldn11-CreERT2-mediated recombination is induced during valve leaflet formation, and efficient labeling of valve endothelial cells was observed in mature valves. The Cldn11-CreERT2 mouse thus provides a valuable tool for functional studies of valves.
|
|
| 16. |
- Panara, Virginia
(författare)
-
The elephant in the cell : A multifaceted undertaking to uncover the molecular principles behind lymphatic endothelial cell identity
- 2023
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The lymphatic vascular network is composed of a series of blind ended vessels, and is involved in physiological functions such as fluid homeostasis, lipid metabolism and immune trafficking. Despite lymphatic endothelial cells being derived from multiple organs, they share common molecular denominators, including the expression of the transcription factor Prox1. However, many of the molecular mechanisms involved in the acquisition and maintenance of lymphatic identity remains to be uncovered.The aim of my thesis is to investigate how transcription factors and chromatin re-arrangements are involved in the acquisition and maintenance of lymphatic identity. In Paper I, the role of the transcription factor mafbb and its ability to partially compensate for the loss of its paralog mafba is investigated, leading to the description of topologically distinct requitements for LECs development in zebrafish. In Paper II, an evolutionary conserved prox1a enhancer is shown to be necessary for correct lymphangiogenesis in mouse, and its ablation is shown to cause the transition of LECs to hematopoietic-like cells. In Paper III, the effects of partial and total prox1 loss in zebrafish are investigated from a transcriptomic and epigenetic perspective, revealing an important role for prox1 in establishing LEC identity by suppressing genes associated with VEC fate. In Paper IV, the cis-regulatory landscape of prox1a is characterised identifying a number of lymphatic enhancers driving expression in either all LECs in the embryo or in anatomically distinct subsets of lymphatic vasculature. Finally, in Paper V, the chromatin organisation signature of LECs is explored, finding new candidate genes and enhancers active in the lymphatic endothelium.In summary, my doctoral studies investigated the different levels of regulation of cell identity involved in LECs differentiation. My work focused on chromatin organisation, enhancer activity, transcription factors and differential gene expression to uncover the complex interactions between these separate mechanisms.
|
|
| 17. |
- Petkova, Milena
(författare)
-
Cell-autonomous and paracrine mechanisms underlying Pik3ca-driven vascular malformations
- 2024
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Vascular malformation is a benign overgrowth of blood or lymphatic vessels leading to life-threatening consequences for affected patients. Activating mutations in the TIE2 receptor cause the majority of venous malformations (VMs), while somatic activating mutations in PIK3CA, leading to the overactivation of the PI3K-AKT pathway, cause both VMs and lymphatic malformations (LMs). Although molecular inhibitors targeting the PI3K-AKT-mTOR pathway, such as rapamycin, have shown beneficial effects, they are not curative. This thesis aimed to explore the endothelial cell-autonomous and paracrine mechanisms underlying Pik3ca-driven pathological vascular growth to identify a rationale for improved and curative therapies for vascular malformations.In Paper I, we reported that one of the most common causative mutations, PIK3CAH1047R, gives rise to two distinct LM subtypes known as macrocystic and microcystic LM in humans. Using a transgenic mouse model with temporally controlled LEC-specific activation of Pik3caH1047R, we found that the growth of microcystic LM is dependent on both the upstream pro-lymphangiogenic VEGF-C-VEGFR3 and the downstream AKT-mTOR signalling. Combination treatment targeting both signalling pathways led to effective inhibition of lesion growth in mice, suggesting a novel therapeutic approach for LM patients. In Paper II, we explored further the endothelial cell-autonomous and paracrine mechanisms underlying microcystic LM growth in mice. Using single-cell RNA sequencing, we identified a new immune-interacting subtype of dermal lymphatic capillary endothelial cells, termed iLECs. We showed that in Pik3ca mutant mice, iLECs produce factors that recruit pro-lymphangiogenic VEGF-C-producing macrophages. Macrophage depletion, inhibition of their recruitment, and anti-inflammatory COX-2 treatment resulted in decreased lymphatic growth, indicating a critical role of paracrine signalling between iLECs and immune cells in the pathogenesis of microcystic LM. In Paper III, we described distinct lymphatic vessel responses to oncogenic PI3K activation in different organs. We observed that while lymphatic vessels in the skin form microcystic LM through vessel sprouting, in certain other organs, they form large cysts reminiscent of macrocystic LM. Finally, we used mice with a BEC-specific activation of Pik3caH1047R to compare disease mechanisms in VM to those in LM in Paper II and to focus further on the former in Paper IV.
|
|
| 18. |
- Petkova, Milena, et al.
(författare)
-
Immune-interacting lymphatic endothelial subtype at capillary terminals drives lymphatic malformation
- 2023
-
Ingår i: The Journal of experimental medicine. - : Rockefeller University Press. - 1540-9538 .- 0022-1007. ; 220:4
-
Tidskriftsartikel (refereegranskat)abstract
- Oncogenic mutations in PIK3CA, encoding p110α-PI3K, are a common cause of venous and lymphatic malformations. Vessel type–specific disease pathogenesis is poorly understood, hampering development of efficient therapies. Here, we reveal a new immune-interacting subtype of Ptx3-positive dermal lymphatic capillary endothelial cells (iLECs) that recruit pro-lymphangiogenic macrophages to promote progressive lymphatic overgrowth. Mouse model of Pik3caH1047R-driven vascular malformations showed that proliferation was induced in both venous and lymphatic ECs but sustained selectively in LECs of advanced lesions. Single-cell transcriptomics identified the iLEC population, residing at lymphatic capillary terminals of normal vasculature, that was expanded in Pik3caH1047R mice. Expression of pro-inflammatory genes, including monocyte/macrophage chemokine Ccl2, in Pik3caH1047R-iLECs was associated with recruitment of VEGF-C–producing macrophages. Macrophage depletion, CCL2 blockade, or anti-inflammatory COX-2 inhibition limited Pik3caH1047R-driven lymphangiogenesis. Thus, targeting the paracrine crosstalk involving iLECs and macrophages provides a new therapeutic opportunity for lymphatic malformations. Identification of iLECs further indicates that peripheral lymphatic vessels not only respond to but also actively orchestrate inflammatory processes.
|
|
| 19. |
- Petkova, Milena, et al.
(författare)
-
Lymphatic malformations : mechanistic insights and evolving therapeutic frontiers
- 2024
-
Ingår i: Journal of Clinical Investigation. - : American Society For Clinical Investigation. - 0021-9738 .- 1558-8238. ; 134:6
-
Tidskriftsartikel (refereegranskat)abstract
- The lymphatic vascular system is gaining recognition for its multifaceted role and broad pathological significance. Once perceived as a mere conduit for interstitial fluid and immune cell transport, recent research has unveiled its active involvement in critical physiological processes and common diseases, including inflammation, autoimmune diseases, and atherosclerosis. Consequently, abnormal development or functionality of lymphatic vessels can result in serious health complications. Here, we discuss lymphatic malformations (LMs), which are localized lesions that manifest as fluid -filled cysts or extensive infiltrative lymphatic vessel overgrowth, often associated with debilitating, even life -threatening, consequences. Genetic causes of LMs have been uncovered, and several promising drug -based therapies are currently under investigation and will be discussed.
|
|
| 20. |
- Pietilä, Riikka, et al.
(författare)
-
Molecular anatomy of adult mouse leptomeninges
- 2023
-
Ingår i: Neuron. - : Elsevier. - 0896-6273 .- 1097-4199. ; 111:23
-
Tidskriftsartikel (refereegranskat)abstract
- Leptomeninges, consisting of the pia mater and arachnoid, form a connective tissue investment and barrier enclosure of the brain. The exact nature of leptomeningeal cells has long been debated. In this study, we iden-tify five molecularly distinct fibroblast-like transcriptomes in cerebral leptomeninges; link them to anatomically distinct cell types of the pia, inner arachnoid, outer arachnoid barrier, and dural border layer; and contrast them to a sixth fibroblast-like transcriptome present in the choroid plexus and median eminence. Newly identified transcriptional markers enabled molecular characterization of cell types responsible for adherence of arach-noid layers to one another and for the arachnoid barrier. These markers also proved useful in identifying the molecular features of leptomeningeal development, injury, and repair that were preserved or changed after traumatic brain injury. Together, the findings highlight the value of identifying fibroblast transcriptional subsets and their cellular locations toward advancing the understanding of leptomeningeal physiology and pathology.
|
|
| 21. |
- Singhal, Dhruv, et al.
(författare)
-
Mapping the lymphatic system across body scales and expertise domains : A report from the 2021 National Heart, Lung, and Blood Institute workshop at the Boston Lymphatic Symposium
- 2023
-
Ingår i: Frontiers in Physiology. - : Frontiers Media S.A.. - 1664-042X. ; 14
-
Forskningsöversikt (refereegranskat)abstract
- Enhancing our understanding of lymphatic anatomy from the microscopic to the anatomical scale is essential to discern how the structure and function of the lymphatic system interacts with different tissues and organs within the body and contributes to health and disease. The knowledge of molecular aspects of the lymphatic network is fundamental to understand the mechanisms of disease progression and prevention. Recent advances in mapping components of the lymphatic system using state of the art single cell technologies, the identification of novel biomarkers, new clinical imaging efforts, and computational tools which attempt to identify connections between these diverse technologies hold the potential to catalyze new strategies to address lymphatic diseases such as lymphedema and lipedema. This manuscript summarizes current knowledge of the lymphatic system and identifies prevailing challenges and opportunities to advance the field of lymphatic research as discussed by the experts in the workshop.
|
|
| 22. |
- Stritt, Simon, et al.
(författare)
-
APOLD1 loss causes endothelial dysfunction involving cell junctions, cytoskeletal architecture, and Weibel-Palade bodies, while disrupting hemostasis
- 2023
-
Ingår i: Haematologica. - : Ferrata Storti Foundation. - 0390-6078 .- 1592-8721. ; 108:3, s. 772-784
-
Tidskriftsartikel (refereegranskat)abstract
- Vascular homeostasis is impaired in various diseases thereby contributing to the progression of their underlying pathologies. The endothelial immediate early gene Apolipoprotein L domain-containing 1 (APOLD1) helps to regulate endothelial function. However, its precise role in endothelial cell biology remains unclear. We have localized APOLD1 to endothelial cell contacts and to Weibel-Palade bodies (WPB) where it associates with von Willebrand factor (VWF) tubules. Silencing of APOLD1 in primary human endothelial cells disrupted the cell junction-cytoskeletal interface, thereby altering endothelial permeability accompanied by spontaneous release of WPB contents. This resulted in an increased presence of WPB cargoes, notably VWF and angiopoietin-2 in the extracellular medium. Autophagy flux, previously recognized as an essential mechanism for the regulated release of WPB, was impaired in the absence of APOLD1. In addition, we report APOLD1 as a candidate gene for a novel inherited bleeding disorder across three generations of a large family in which an atypical bleeding diathesis was associated with episodic impaired microcirculation. A dominant heterozygous nonsense APOLD1:p.R49* variant segregated to affected family members. Compromised vascular integrity resulting from an excess of plasma angiopoietin-2, and locally impaired availability of VWF may explain the unusual clinical profile of APOLD1:p.R49* patients. In summary, our findings identify APOLD1 as an important regulator of vascular homeostasis and raise the need to consider testing of endothelial cell function in patients with inherited bleeding disorders without apparent platelet or coagulation defects.
|
|
| 23. |
- Stritt, Simon, et al.
(författare)
-
Homeostatic maintenance of the lymphatic vasculature
- 2021
-
Ingår i: Trends in Molecular Medicine. - : Elsevier. - 1471-4914 .- 1471-499X. ; 27:10, s. 955-970
-
Tidskriftsartikel (refereegranskat)abstract
- The lymphatic vasculature is emerging as a multifaceted regulator of tissue homeostasis and regeneration. Lymphatic vessels drain fluid, macromolecules, and immune cells from peripheral tissues to lymph nodes (LNs) and the systemic circulation. Their recently uncovered functions extend beyond drainage and include direct modulation of adaptive immunity and paracrine regulation of organ growth. The developmental mechanisms controlling lymphatic vessel growth have been described with increasing precision. It is less clear how the essential functional features of lymphatic vessels are established and maintained. We discuss the mechanisms that maintain lymphatic vessel integrity in adult tissues and control vessel repair and regeneration. This knowledge is crucial for understanding the pathological vessel changes that contribute to disease, and provides an opportunity for therapy development.
|
|
| 24. |
- Zhang, Yan, et al.
(författare)
-
Cdh5-lineage–independent origin of dermal lymphatics shown by temporally restricted lineage tracing
- 2022
-
Ingår i: Life Science Alliance. - : Life Science Alliance, LLC. - 2575-1077. ; 5:11
-
Tidskriftsartikel (refereegranskat)abstract
- The developmental origins of lymphatic endothelial cells (LECs) have been under intense research after a century-long debate. Although previously thought to be of solely venous endothelial origin, additional sources of LECs were recently identified in multiple tissues in mice. Here, we investigated the regional differences in the origin(s) of the dermal lymphatic vasculature by lineage tracing using the pan-endothelial Cdh5-CreERT2 line. Tamoxifen-induced labeling of blood ECs at E9.5, before initiation of lymphatic development, traced most of the dermal LECs but with lower efficiency in the lumbar compared with the cervical skin. By contrast, when used at E9.5 but not at E11.5, 4-hydroxytamoxifen, the active metabolite of tamoxifen that provides a tighter window of Cre activity, revealed low labeling frequency of LECs, and lymphvasculogenic clusters in the lumbar skin in particular. Temporally restricted lineage tracing thus reveals contribution of LECs of Cdh5-lineage–independent origin to dermal lymphatic vasculature. Our results further highlight Cre induction strategy as a critical parameter in defining the temporal window for stage-specific lineage tracing during early developmental stages of rapid tissue differentiation.
|
|
