Angiogenesis

NRP1 is a therapeutic target for inhibiting vascular endothelial growth factor (VEGF)-induced blood vessel dysfunction. The small molecule EG00229 was designed to inhibit VEGF binding to NRP1 and reduce pathological blood vessel growth. However, it is unknown whether EG00229 could also be used to reduce VEGF164-induced vascular leakage, which often exacerbates ischemic diseases due to VEGF upregulation. Here, we show that prior treatment with EG00229 prevents VEGF164-induced vascular permeability signalling, but, unexpectedly, also find that EG00229 increased rather than inhibited vascular leakage. Thus, EG00229 increased vascular leakage either when added alone or concurrently with VEGF164, both in perfused retinal explants and across primary brain EC monolayers. This EG00229-induced vascular leakage was not an off-target effect, because it relied on endothelial NRP1 expression and NRP1s VEGF164 binding pocket, yet was independent of VEGFR1 and VEGFR2. Moreover, EG00229 activated molecular events typical of VEGF164-induced paracellular permeability, including p38 MAP kinase (p38) and SRC family kinase (SFK) phosphorylation as well as CDH5 rearrangement in endothelial junctions. Investigating EG00229-induced signalling therefore helps elucidate NRP1-dependent mechanisms of paracellular permeability induction and might help identifying new approaches to modulate the neurovascular barrier.

RationaleMutation in human arteriovenous malformation (AVM) causative genes in a fraction of endothelial cells (ECs) causes AVMs in mice. It is unclear how a small number of mutant ECs can lead to AVM formation. ObjectiveTo understand how a fraction of mutant ECs causes AVM, we tested the following hypotheses: (1) activin receptor-like kinase 1 (Alk1 or Acvlr1) mutant brain ECs undergo clonal expansion upon angiogenic stimulation, (2) Alk1 mutant ECs display growth advantage, (3) the burden of Alk1 mutant ECs correlates with AVM severity, and (4) Alk1 mutant bone marrow (BM) derived ECs alone is sufficient to cause AVM. Methods and ResultsWe used PdgfbiCreER;Alk1f/f;confetti+/- mice which express an EC-specific tamoxifen (TM)-inducible Cre recombinase, a Cre-regulated confetti transgene, and Alk1 floxed alleles. Brain AVMs were induced by direct brain injection of an adeno-associated viral vector expressing vascular endothelial growth factor (AAV-VEGF) followed with intra-peritoneal injection of TM two weeks later. Color-predominance of confetti reporter in AVMs compared to control brain ECs suggested that clonal expansion was associated with AVM development. We treated PdgfbiCreER;Alk1f/f with different doses of TM to create a mosaic of wild-type (WT) and mutant ECs and found that equal numbers of Alk1+ and Alk1- ECs were proliferating. Increase of TM dose increased the number of Alk1- ECs, the abnormal vessels in brain AVMs, the number of arteriovenous shunts in the intestines, and mouse mortality. To test if mutation of Alk1 in BM-derived ECs can cause brain AVM, we transplanted WT mice with BM of PdgfbiCreER;Alk1f/f mice. After AAV-VEGF and TM treatment, these mice developed AVMs in their brains and arteriovenous shunts in their intestines. ConclusionClonal expansion of Alk1 mutant ECs could partly explain why a fraction of mutant ECs causes AVM. Mutation of AVM causal genes in BM-derived ECs is sufficient to cause AVM formation.

Hyaloid vascular regression is a critical developmental process essential for vitreous transparency and normal vision, yet the molecular cues orchestrating its involution remain incompletely defined. Here, we identify Notch1 as a pivotal regulator of hyaloid vessel clearance, acting independently of apoptosis to coordinate endothelial detachment, transient plasticity, and migration. Using an endothelial-specific Notch1 knockout mouse model, we demonstrate that loss of Notch1 results in persistent hyaloid vasculature characterized by excessive proliferation and stabilization of the vascular network. Mechanistically, Notch1 activation during the regression window induces endothelial-to-mesenchymal transition (EndoMT) marked by Snail1 and Slug upregulation. This transcriptional signature is accompanied by detachment of endothelial cells from the vascular tubes. In contrast, Notch1-deficient hyaloid vessels retain endothelial cells stably adherent to the vessel wall. Further analysis reveals that Wnt receptors FZD4, LRP5 and LRP6 previously implicated in hyaloid involution are transcriptionally downregulated in Notch1-deficient hyaloids, suggesting that the collaboration between these processes may occur through crosstalk between the Notch and Wnt pathways. Collectively, our findings uncover a Notch1-driven multicellular regression program that governs developmental vessel regression, redefining the molecular principles of vascular pruning. These results have broad implications for understanding vascular remodeling in both physiological and pathological contexts and may guide therapeutic strategies to modulate vascular regression in ocular disorders. One-Sentence SummaryNotch1 drives hyaloid regression through a multicellular program that defines an apoptosis-non-exclusive paradigm of vessel pruning.

Despite the absolute requirement of Delta/Notch signaling to activate lateral inhibition during early blood vessel development, many mechanisms remain unclear. Here, we identify EHD2 and EHBP1 as novel regulators of Notch activation in endothelial cells through controlling endocytosis of Delta-like ligand 4 (Dll4). Knockout of EHBP1 and EHD2 in zebrafish produced a significant increase in ectopic sprouts in zebrafish intersomitic vessels during development and a reduction in downstream Notch signaling. In vitro, EHBP1 and EHD2 localized to plasma membrane-bound Dll4 and actin independently of clathrin. Disruption of caveolin endocytosis resulted in EHBP1 and EHD2 failing to organize around Dll4 as well as loss of Dll4 internalization in endothelial cells. Overall, we demonstrate that EHBP1 and EHD2 regulate Dll4 endocytosis by anchoring caveolar endocytic pits to the actin cytoskeleton.

New blood vessel formation, or angiogenesis, is characteristic of chronic diseases such as cancer, rheumatoid arthritis and vision-threatening conditions. Vascular Endothelial growth factor (VEGFA) and its receptor VEGFR2 drive neovascularization and hyperpermeability in these pathologies. One consequence of VEGFR2 activation is decreased stability of endothelial cell (EC) junctions through internalization of VE-Cadherin, allowing re-arrangement of sprouting ECs. Evidence suggests roles for heparan sulfate proteoglycans in angiogenesis and we show that Syndecan-4 (SDC4) expression is upregulated during pathological angiogenesis and is required for efficient VE-Cadherin internalization. Angiogenic responses in both tumor and neovascular eye disease models are impaired in Syndecan-4 null mice (Sdc4-/-), as is dermal hyper-permeability response to VEGFA. We show SDC4 resides at EC junctions and interacts with VE-Cadherin, an association lost upon VEGFA-stimulation, and this is SDC4 phosphorylation-dependent. Finally, we show that pathological angiogenic responses can be inhibited in a model of age-related macular degeneration by targeting SDC4. This study identifies SDC4 as a key component of VE-Cadherin trafficking and, as such, a critical regulator of pathological angiogenesis and vascular permeability.

Endothelial cells engage extracellular matrix and basement membrane components through integrin-mediated adhesion to promote angiogenesis. Our previous studies demonstrated that endothelial expression of laminin-411 and laminin-511 as well as 6 integrins is required for endothelial sprouting and tube formation in organotypic angiogenesis assays. These studies demonstrated that 6 integrins promote migration and regulate the expression of ANGPT2 and CXCR4 and that 6-dependent regulation of CXCR4 contributes to endothelial morphogenesis in our assays. However, these studies did not identify specific roles for the 6{beta}1, 6{beta}4, or 3{beta}1 laminin-binding integrins. Here, we employ RNAi technology to parse the contributions of these integrins. We demonstrate that 6{beta}4 promotes migration, sprouting, and tube formation, and also positively regulates the expression of ANGPT2, but does not promote CXCR4 expression, suggesting that 6{beta}1 functions in this regulation. Additionally, we show that 3{beta}1 regulates endothelial sprouting and tube formation, but is not required for migration in our assays or for the expression of ANGPT2 or CXCR4. Integrin 3{beta}1 promotes the expression of NRP1 and ID1 RNAs, both of which are known to promote angiogenesis. Taken together, our results indicate that laminin-binding integrins play distinct roles during endothelial morphogenesis and do not compensate for one another in organotypic culture. Summary StatementThe laminin-binding integrins 3{beta}1, 6{beta}1, and 6{beta}4 contribute to endothelial sprouting and tube formation in organotypic angiogenesis assays.

RationaleHereditary haemorrhagic telangiectasia (HHT) is an inherited bleeding disorder characterised by arteriovenous malformations (AVMs). Such AVMs affect lungs, liver and brain, whilst telangiectases in mucocutaneous tissues are prone to haemorrhage. HHT type I is caused by loss-of-function endoglin (ENG) mutations. Evidence suggests AVMs result from abnormal responses to VEGF signalling. ObjectiveWe therefore characterised the vascular abnormalities in eng mutant zebrafish and investigated whether these are prevented by inhibiting different pathways downstream of VEGF signalling. Methods and ResultsWe used light sheet fluorescence microscopy to visualise the vasculature in engmu130 mutant zebrafish. In addition to previously described significantly enlarged dorsal aorta and posterior cardinal vein at 3d post fertilisation, engmu130 embryos had an enlarged basilar artery (BA), and increased formation of endothelial "kugeln" on cerebral vessels. Adult engmu130 fish developed skin AVMs, retinal vascular abnormalities, and an enlarged heart. Tivozanib (AV951), a VEGF receptor tyrosine kinase inhibitor, prevented development of the abnormally enlarged major vessels and normalised the number of kugeln in engmu130 embryos. Inhibiting discrete signalling pathways downstream of VEGFR2 in engmu130 embryos gave further insights. Inhibiting TOR or MEK prevented the abnormal trunk and cerebral vasculature phenotype, whilst targeting NOS and MAPK had no effect. Combining subtherapeutic TOR and MEK inhibition prevented the vascular phenotype, suggesting synergy between TOR and MEK/ERK signalling pathways. ConclusionsThese results indicate the HHT-like phenotype in zebrafish endoglin mutants can be mitigated through modulation of VEGF signalling, and implicate combination low dose ERK and TOR pathway inhibitors as a therapeutic strategy in HHT. Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

BACKGROUNDHereditary hemorrhagic telangiectasia (HHT) is a genetic disorder caused by pathogenic variants in the endothelial TGF{beta}/BMP pathway, crucial for the vascular arterial-venous differentiation. Vascular defects result in fragile and malformed vessels. The precise mechanisms driving vascular network failure remain incompletely understood, complicating the design of targeted therapies. METHODSNasal telangiectasias from HHT patients carrying variants in ACVRL1 or ENG were used to perform scRNA-seq (2 ACVRL1- and 1 ENG-patient) and spatial transcriptomics (1 ACVRL1 and 1 ENG) to uncover endothelial cells (EC) populations. Vascular characteristics within biopsies were evaluated using transmission electron microscopy (TEM) (1 ACVRL1 and 1 ENG) and histological analyses (23 ACVRL1 and 7 ENG), with particular attention to regions exhibiting varying degrees of damage. RESULTSComparing our HHT tissues with healthy donor from the literature, we identified cellular heterogeneity within EC populations, revealing two distinct venous clusters: a stable, quiescent population (Mature Vein) and an activated, pro-inflammatory population (HHT Vein). The coexistence of these two clusters suggests cellular diversity within the biopsy, further validated by TEM and histology, revealing a juxtaposition of well-organized collagen and cellular architecture with severely disrupted, fibrotic regions. Moreover, cellular crosstalk analyses allowed us to identify critical ligands in ECs that interact with fibroblasts and mural cells. In particular, we found Midkine (MDK) lost in HHT Vein ECs with further validation in vitro, suggesting its potential role in cellular stability. Furthermore, spatial transcriptomics allowed to further uncover pathologic phenotypes in cells neighboring HHT Vein ECs. CONCLUSIONSHHT biopsies exhibit localized inflamed and fibrotic vascular areas with the presence of different transcriptional sub-populations of EC. Within the same tissue, stable and activated ECs can be distinguished. The pathologic-like EC cluster, present exclusively in the HHT samples, may contribute to vascular leakage through the loss of important ligands involved in cellular communication.

Neuropilin (NRP) expression is highly correlated with poor outcome in multiple cancer subtypes. As known co-receptors for vascular endothelial growth factor receptors (VEGFRs), core drivers of angiogenesis, past investigations have alluded to their functional roles in facilitating tumorigenesis by promoting invasive vessel growth. Despite this, it remains unclear as to whether NRP1 and NRP2 act in a synergistic manner to enhance pathological angiogenesis. Here we demonstrate, using NRP1ECKO, NRP2ECKO and NRP1/NRP2ECKO mouse models, that maximum inhibition of primary tumour development and angiogenesis is only achieved when both endothelial NRP1 and NRP2 are targeted simultaneously. Metastasis and secondary site angiogenesis were also significantly inhibited in NRP1/NRP2ECKO animals. Mechanistic studies revealed that co-depleting NRP1 and NRP2 in mouse-microvascular endothelial cells (ECs) stimulates rapid shuttling of VEGFR-2 to Rab7+ endosomes for proteosomal degradation. Our results highlight the importance of targeting both NRP1 and NRP2 to modulate tumour angiogenesis.

BackgroundSporadic venous malformation (VM) and angiomatosis of soft tissue (AST) are benign, congenital vascular anomalies affecting venous vasculature. Depending on the size and location of the lesion, symptoms vary from motility disturbances to pain and disfigurement. Due to high recurrence of the lesions more effective therapies are needed. MethodsAs targeting stromal cells has been an emerging concept in anti-angiogenic therapies, here, by using VM/AST patient samples, RNA-sequencing, cell culture techniques and a xenograft mouse model, we investigated the crosstalk of endothelial cells (EC) and fibroblasts and its effect on vascular lesion growth. ResultsWe report, for the first time, expression and secretion of transforming growth factor A (TGFA) in ECs or intervascular stromal cells in AST and VM lesions. TGFA induced secretion of VEGF-A paracrinally, and regulated EC proliferation. Oncogenic PIK3CA variant in p.H1047R, a common somatic mutation found in these lesions, increased TGFA expression, enrichment of hallmark hypoxia, and in a mouse xenograft model, lesion size and vascularization. Treatment with afatinib, a pan-ErbB tyrosine-kinase inhibitor, decreased vascularization and lesion size in mouse xenograft model with ECs expressing oncogenic PIK3CA p.H1047R variant and fibroblasts. ConclusionsBased on the data, we suggest that targeting of both intervascular stromal cells and ECs is a potential treatment strategy for vascular lesions having a fibrous component. FundingAcademy of Finland, Ella and Georg Ehnrooth foundation, the ERC grants, Sigrid Juselius Foundation, Finnish Foundation for Cardiovascular Research, Jane and Aatos Erkko Foundation, and Department of Musculosceletal and Plastic Surgery, Helsinki University Hospital. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/509204v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@14ec94aorg.highwire.dtl.DTLVardef@1812747org.highwire.dtl.DTLVardef@39d01eorg.highwire.dtl.DTLVardef@18e9e35_HPS_FORMAT_FIGEXP M_FIG Graphical abstract. Proposed model for the paracrine signaling of TGFA/VEGF-A in vascular lesion Schematic illustration showing the general structure of venous malformation or angiomatosis of soft tissue. Pathological vasculature in the lesion (dark blue) is surrounded by disorganized extracellular matrix (ECM) and intervascular stromal cells (SCs, orange). High magnification from the area close to vessel wall demonstrates the proposed model for crosstalk between endothelial cells (ECs) and SCs. A mutation in phosphatidylinositol-4,5-biphosphate 3-kinase catalytic subunit alpha (PIK3CA) gene (1) or other processes promote ECs to express high level of transforming growth factor A (TGFA) (2). TGFA binds to epithelial growth factor receptor (EGFR) on the surface of adjacent SCs (3). Activated EGFR-downstream signaling (4) promotes elevated expression of vascular endothelial growth factor (VEGF)-A in SCs and increases the expression of TGFA (5). VEGF-A secreted from SCs (6) binds to VEGF-recetor-2 (VEGFR2) on surface of ECs (7) and together with TGFA activates angiogenic EC phenotype. TGFA secreted from the SCs (8), can further activate EGFR and its downstream signaling. C_FIG

The mechanosensing properties of endothelial cell-cell junctions are essential for vascular beds to respond to the mechanical forces exerted by blood flow. In states of disturbed flow, endothelial cells (ECs) become activated and transition to a pro-inflammatory, atheroprone phenotype. Here, we investigated the role of transmembrane glycoprotein neuropilin 2 (NRP2) in maintaining adherens junction integrity using cultured immortalised mouse ECs and a genetically modified mouse model to demonstrate the effects of an endothelial-specific deletion of Nrp2 in vivo. We reveal that, akin to its ortholog, Nrp1, Nrp2 exists as a constituent of adherens junctions, maintaining surface availability of VE cadherin by promoting its interaction with p120 catenin. As a consequence, endothelial knockout mice (Nrp2flfl.ECKO) display hyperpermeable retinal vasculature during development. Nrp2 depletion was subsequently found to activate key pro-inflammatory cytokines and adhesion molecules known to participate in the progression of atherogenesis, in addition to increased immune cell attachment aortic plaque development. These findings describe a role for Nrp2 in maintaining junctional signalling in ECs, protecting against endothelial activation during a state of vascular disease.

During vascular development, arteries and veins form in a stepwise process that combines vasculogenesis and sprouting angiogenesis. Despite extensive data on the mechanisms governing blood vessel assembly at the single cell level, little is known about how cell populations migrate in a finely tuned and coordinated manner, and distribute precisely between arteries and veins. Here, we use an endothelial-specific zebrafish reporter, arteriobow, to label small cohorts of arterial cells and trace their progeny from the initial events of vasculogenesis through the process of arterio-venous remodeling. We reveal that the genesis of both arteries and veins relies on the coordination of ten types of collective cell behaviors originating from discrete endothelial cell clusters. Within these behavioral categories, we identify a heterogeneity of collective cell dynamics specific to either arterial or venous remodeling. Using pharmacological blockade, we further show that factors known to control vascular patterning such as cell-intrinsic Notch signaling and cell-extrinsic blood flow, potentially act as regulators by coordinating endothelial cohorts behavior, which in turn instructs the future territory of arterio-venous remodeling.

AbstractHereditary Hemorrhagic Telangiectasia (HHT) is a genetic vascular disorder characterized by distinct vascular malformations, including deep organ arteriovenous malformations (AVMs) and mucocutaneous telangiectasias. People with HHT inherit monoallelic pathogenic variants in members of the TGF{beta} signaling cascade (ACVRL1, ENG and SMAD4), resulting in a loss of gene function and dysangiogenesis. While these heterozygous inactivating mutations are present in all cells, malformations develop locally, indicating a focal trigger of onset. Indeed, recent human sequencing studies revealed that second-hit somatic mutations, resulting in complete bi-allelic loss of gene function, are linked to lesion formation in the three major types of HHT (HHT1, HHT2, JP/HHT). To model the loss of heterozygosity (LOH) associated with HHT patients, we generated new Eng and Smad4 HHT mouse models whereby endothelial cell-specific, somatic LOH mutations are induced within a heterozygous loss of function background (HHT-iEC-LOH). The HHT-iEC-LOH models recapitulate the mosaic makeup of patient malformations and indicate that multiple, distinct secondary somatic mutations can contribute to AVM onset. Utilizing immunofluorescent staining, blue latex vasculature casting, weighted tracer perfusions, and lineage tracing studies, HHT-iEC-LOH models were phenotypically assessed and compared to traditional inducible endothelial cell knockout (HHT-iECKO) HHT models. Overall, HHT-iEC-LOH mice exhibit increased malformation frequency and vascular phenotypes that are comparable or exceed the severity of iECKO models. Significantly, HHT-iEC-LOH mice can be induced early in development and live into adulthood, displaying persistent cerebrovascular phenotypes. The heightened patient representation offered by these newly developed models enables the study of long-term disease progression and testing of therapeutic interventions.

Integrin trafficking to and from membrane adhesions is a crucial mechanism that dictates many aspects of a cells behaviour, including motility, polarisation, and invasion. In endothelial cells (ECs), the intracellular traffic of 5 integrin is regulated by both neuropilin 1 (NRP1) and neuropilin 2 (NRP2), yet the redundancies in function between these co-receptors remain unclear. Moreover, the endocytic complexes that participate in NRP-directed traffic remain poorly annotated. Using label-free quantitative mass spectrometry of 5 integrin associations in ECs we identify 5 trafficking pathways that depend on NRP1, NRP2, or both NRPs. We identify a trafficking pathway that depends on both NRPs: one that impinges on the GTPase-activating protein p120RasGAP. This pathway promotes the recycling of 5 integrin from early endosomes. Mechanistically, p120RasGAP enables transit of endocytosed 5 integrin-NRP1-NRP2 complexes to Rab11+ recycling endosomes, promoting cell polarisation and fibronectin (FN) fibrillogenesis. Silencing of both NRP receptors, or p120RasGAP, results in the accumulation of 5 integrin in early endosomes, a loss of 5 integrin from surface adhesions, and attenuated EC polarisation. Importantly, endothelial-specific deletion of both NRP1 and NRP2 in the postnatal retina recapitulated our in vitro findings, severely impairing FN fibrillogenesis and polarised sprouting. Our data assign an essential role for p120RasGAP during integrin traffic in ECs and support a hypothesis that NRP receptors can co-traffic internalised cargoes.

Clathrin-mediated endocytosis (CME) is a process vital to angiogenesis as well as general vascular homeostasis. In pathologies where supraphysiological growth factor signaling underlies disease etiology, such as in diabetic retinopathy and solid tumors, strategies to limit chronic growth factor signaling by way of CME have been shown to have tremendous clinical value. ADP ribosylation factor 6 (Arf6) is a small GTPase that promotes the assembly of actin necessary for CME. In its absence, growth factor signaling is greatly diminished, which has been shown to ameliorate pathological signaling input in diseased vasculature. However, it is less clear if there are bystander effects related to loss of Arf6 on angiogenic behaviors. Our goal was to provide a analysis of Arf6s function in angiogenic endothelium, focusing on its role in lumenogenesis as well as its relation to actin and CME. We found that Arf6 localized to both filamentous actin and sites of CME in 2-dimensional culture. Loss of Arf6 distorted both apicobasal polarity and reduced the total cellular filamentous actin content, and this may be the primary driver underlying gross dysmorphogenesis during angiogenic sprouting in its absence. Our findings highlight that endothelial Arf6 is a potent mediator of both actin regulation and CME.

Uveal melanoma is an aggressive intraocular tumour characterised by a limited number of genetic alterations. However, the evolution of this malignancy remains enigmatic. In this study, we performed a deep quantitative analysis of 80 primary uveal melanomas by novel digital PCR-based approaches. Mutations were quantified by targeted and drop-off mutation assays, copy number alterations were precisely measured by quantifying the allelic imbalance of heterozygous single-nucleotide polymorphisms. By comparing the absolute abundances of genetic alterations present in a bulk tumour, the heterogeneity and early evolution could be inferred. Tumour progression was further studied by analysing matched primary and metastatic lesions from five patients. Gq signalling mutations were generically and always clonally present, suggesting to be acquired in the earliest stage of uveal melanoma development ( primary driver). Next, three main evolutionary subtypes could be identified based on having an EIF1AX mutation, SF3B1 mutation or monosomy 3p. These alterations were usually mutually-exclusive and (near-) clonally abundant, suggesting to represent distinct secondary drivers. This contrasts with gains and amplifications of chromosome 8q, which were not restricted to one of the main subtypes and showed subclonality in 31% of the affected tumours. These tertiary alterations were not required for metastatic dissemination. Using high-resolution analyses, we identified systematic differences in the evolutionary timing of genetic events in uveal melanoma. The observed intratumour heterogeneity suggests a more complex model of gradual tumour evolution and argues for a comprehensive genetic analysis in clinical practice, which may be facilitated by the sensitive digital PCR assays developed in this study.

Capillary malformations (CM) are congenital vascular irregularities of capillary and venous blood vessels that appear in the skin, leptomeninges of the brain, and the choroid of the eye in the disorder known as Sturge Weber Syndrome (SWS). More common are non-syndromic CM found only in the skin, without brain or ocular involvement. A somatic activating mutation in GNAQ (p.R183Q) is found in [~]90% of syndromic and non-syndromic CM specimens and is present in CD31pos endothelial cells isolated from brain and skin CM specimens. Endothelial expression of the GNAQ p.R183Q variant is sufficient to form CM-like vessels in mice. Given the distinct features and functions of blood vessels in the brain versus the skin, we examined the features of CM vessels in both tissues to gain insights into the pathogenesis of CM. Herein, we present morphologic characteristics of CM observed in specimen from brain and skin. The GNAQ p.R183Q variant allelic frequency in each specimen was determined by droplet digital PCR. Sections were stained for endothelial cells, tight junctions, mural cells, and macrophages to assess the endothelium as well as perivascular constituents. CM blood vessels in brain and skin were enlarged, exhibited fibrin leakage and reduced zona occludin-1, and were surrounded by MRC1pos/LYVE1pos macrophages. In contrast, the CMs from brain and skin differ in endothelial sprouting activity and localization of mural cells. These characteristics might be helpful in the development of targeted and/or tissue specific therapies to prevent or reverse non-syndromic and syndromic CM. Statements and DeclarationsNone

Pathogenic variants in kinesin KIF11 underlie microcephaly-lymphedema-chorioretinopathy (MLC) syndrome. Although well known for regulating spindle dynamics ensuring successful cell division, the association of KIF11 (encoding EG5) with development of the lymphatic system, and how KIF11 pathogenic variants lead to lymphatic dysfunction and lymphedema remain unknown. Using patient-derived lymphoblastoid cells, we demonstrate that MLC patients carrying pathogenic stop-gain variants in KIF11 have reduced mRNA and protein levels. Lymphoscintigraphy showed reduced tracer absorption, and intestinal lymphangiectasia was detected in one patient, pointing to impairment of lymphatic function caused by KIF11 haploinsufficiency. We reveal that KIF11 is expressed in early human and mouse development with the lymphatic markers VEGFR3, Podoplanin and PROX1. In zebrafish, scRNA-seq identified KIF11 specifically expressed in endothelial precursors. In human lymphatic endothelial cells (LECs), EG5 inhibition with Ispinesib, reduces VEGFC-driven AKT phosphorylation, migration and spheroid sprouting. KIF11 knockdown reduces PROX1 and VEGFR3 expression, providing for the first time a link between KIF11 and drivers of lymphangiogenesis and lymphatic identity.

The preferential accumulation of vascular smooth muscle cells on arteries versus veins during early development is a well-described phenomenon, but the molecular pathways underlying this polarization are not well understood. During zebrafish embryogenesis the cxcr4a receptor (mammalian CXCR4) and its ligand cxcl12b (mammalian CXCL12) are both preferentially expressed on arteries at time points consistent with the arrival and differentiation of the first vascular smooth muscle cells (vSMCs). We show that autocrine cxcl12b/cxcr4 activity leads to increased production of the vSMC chemoattractant ligand pdgfb by endothelial cells in vitro and increased expression of pdgfb by arteries in vivo. Additionally, we demonstrate that expression of the well-characterized blood flow-regulated transcription factor klf2a in primitive veins negatively regulates cxcr4/cxcl12 and pdgfb expression, restricting vSMC recruitment to the arterial vasculature. Together, this signaling axis leads to the differential acquisition of smooth muscle cells at sites where klf2a expression is low and both cxcr4a and pdgfb are co-expressed, i.e. arteries during early development.

This study investigates the impact of rapamycin and propranolol on cerebral cavernous malformations (CCMs). Employing an unbiased transcriptomic analysis, we aimed to comprehensively elucidate the molecular mechanisms underlying these drug effects in Mouse Brain Microvascular Endothelial Cells (mBMEC) deficient in Ccm3. While propranolol shows limited efficacy in modulating the CCM transcriptomic phenotype in mBMEC, rapamycin demonstrates a higher impact. Rapamycin reverses gene expression changes induced by Ccm3 deficiency, restoring Klf2/4-dependent genes like Nos3, Adamts1, and Thbs1. Notably, we observed a reduction in KLF2 protein levels in Ccm3 KO cells treated with rapamycin. Critically, in vivo experiments demonstrate that a combination of rapamycin and lapatinib effectively reduces lesion volume in a chronic CCM model. This finding is particularly noteworthy as it suggests a potential treatment strategy for existing lesions. In summary, our work describes a new mechanism for the effects of rapamycin in Ccm3- deficient cells and identifies a new drug combination in the treatment of cavernomas.