Angiogenesis

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.

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.

Focal adhesion kinase (FAK) functions as a signaling and scaffolding protein within endothelial cells (ECs) impacting blood vessel function and tumor growth. Interpretations of EC FAK-null phenotypes are complicated by related PYK2 (protein tyrosine kinase 2) expression, and to test this, we created PYK2-/- FAKfl/fl mice with tamoxifen-inducible EC-specific Cre recombinase expression. At 11 weeks of age, EC FAK inactivation resulted in increased heart and lung mass and vascular leakage only on a PYK2-/- background. Surprisingly, [~]90% of PYK2-/- EC FAK-/- mice survived to 75 weeks of age. Syngeneic melanoma, breast, or lung carcinoma tumors did not grow in PYK2-/- EC FAK-/- mice, but tumors grew normally in PYK2-/- EC FAKfl/fl mice lacking Cre. This tumor inhibitory phenotype was associated with abortive EC vessel sprouting, enhanced EC p53 tumor suppressor and p21CIP1 (cyclin-dependent inhibitor 1) expression, and alterations in serum cytokine levels. To discern the role of FAK kinase versus scaffolding activity in ECs, we generated kinase defective (FAK K454R, KD) PYK2-/- EC FAKfl/KD and PYK2-/- EC FAKfl/WT (WT, wildtype) mice. Hemizygous EC FAK-/KD expression supported primary tumor growth but not metastasis, implicating EC FAK activity in tumor dissemination. In vitro, hemizygous expression of either WT or KD FAK suppressed EC p21CIP1 levels and cell death observed in primary PYK2-/- EC FAK-/- ECs. Combined FAK and PYK2 knockdown in tumor cells also increased p21CIP1 and PARP1 (poly ADP-ribose polymerase 1) levels in a p53-associated manner impacting anchorage-independent growth. Together, these results underscore the linkage between PYK2 and FAK loss with p53 activation impacting tumor growth. Impact StatementPYK2-null combined with endothelial cell-specific FAK transgenic mouse models show that loss of FAK activity limits tumor spread and that genetic or chemical degradation preventing combined FAK-PYK2 expression may be an approach to induce a p53-associated anti-tumor response.

The endothelial glycocalyx, lining the apical surface of the endothelium, is involved in a host of vascular processes. The layer contains a network of membrane-bound proteoglycans and glycoproteins. One such glycoprotein is endomucin (EMCN), which our lab has revealed is a modulator of VEGFR2 function. Intravitreal injection of siEMCN into the eyes of P5 mice impairs vascular development. In vitro silencing of EMCN suppresses VEGF-induced proliferation and migration. Signaling pathways that drive cell migration converge on cytoskeletal remodeling. By coupling co-immunoprecipitation with liquid chromatography/mass spectrometry, we identified interactions between EMCN, and proteins associated with actin cytoskeleton organization. The aim of the study was to investigate the influence of EMCN on cytoskeleton dynamics in angiogenesis. EMCN depletion resulted in reduction of F-actin levels, whereas overexpression of EMCN induced membrane protrusions in cells that were rich in stress fibers. The reorganization of the actin filaments did not depend on VEGFR2 signaling, suggesting that EMCN is a potential connection between the cytoskeleton and the glycocalyx.

The mammalian endothelium which lines all blood vessels responds to soluble factors which control vascular development and sprouting. Endothelial cells bind to vascular endothelial growth factor A via two different receptor tyrosine kinases (VEGFR1, VEGFR2) which regulate such cellular responses. The integration of VEGFR signal transduction and membrane trafficking is not well understood. Here, we used a yeast-based membrane protein screen to identify VEGFR-interacting factor(s) which modulate endothelial cell function. By screening a human endothelial cDNA library, we identified a calcium-binding protein, S100A6, which can interact with either VEGFR. We found that S100A6 binds in a calcium-dependent manner to either VEGFR1 or VEGFR2. S100A6 binding was mapped to the VEGFR2 tyrosine kinase domain. Depletion of S100A6 impacts on VEGF-A-regulated signaling through the canonical mitogen-activated protein kinase (MAPK) pathway. Furthermore, S100A6 depletion caused contrasting effects on biosynthetic VEGFR delivery to the plasma membrane. Co-distribution of S100A6 and VEGFRs on tubular profiles suggest the presence of transport carriers that facilitate VEGFR trafficking. We propose a mechanism whereby S100A6 acts as a calcium-regulated switch which facilitates biosynthetic VEGFR trafficking from the TGN-to-plasma membrane. VEGFR-S100A6 interactions thus enable integration of signaling and trafficking pathways in controlling the endothelial response to VEGF-A.

The interaction of Eph receptor tyrosine kinases with their transmembrane ligands; the ephrins, is important for the regulation of cell-cell communication. Ephrin-Eph signaling is probably best known for the discrimination of arterial and venous territories by repulsion of venous endothelial cells away from those with an arterial fate. Ultimately, cell repulsion is mediated by initiating the collapse of the actin cytoskeleton in membrane protrusions. Here, we investigated the role of the Ena/VASP family of actin binding proteins in endothelial cell repulsion initiated by ephrin ligands. Human endothelial cells dynamically extended sheet-like lamellipodia over ephrin-B2 coated surfaces. While lamellipodia of control siRNA transfected cells rapidly collapsed, resulting in a pronounced cell repulsion from the ephrin-B2 surfaces, the knockdown of Ena/VASP proteins impaired the cytoskeletal collapse of membrane protrusions and the cells no longer avoided the repulsive surfaces. Mechanistically, ephrin-B2 stimulation elicited the EphB-mediated tyrosine phosphorylation of VASP, which abrogated its interaction with the focal adhesion protein Zyxin. Nck2 was identified as a novel VASP binding protein, which only interacted with the tyrosine phosphorylated VASP protein. Nck links Eph-receptors to the actin cytoskeleton. Therefore, we hypothesize that Nck-Ena/VASP complex formation is required for actin reorganization and/or Eph receptor internalization downstream of ephrin-Eph interaction in endothelial cells, with implications for endothelial navigation and pathfinding.

Abstract (formatted)O_ST_ABSBackgroundC_ST_ABSVenous malformation (VM) are developmental defects of the vasculature characterized by tremendously enlarged and dysfunctional veins. Gain-of-function somatic mutations in the endothelial-specific tyrosine kinase receptor TIE2 have been identified as leading driver of VM pathogenesis. The aim of this study was to determine whether the aberrant venous lumen expansion is caused by recruitment of wild-type endothelial cells (EC) to the lesion or by TIE2-mutant EC clonal expansion. MethodsTo investigate the contribution of TIE2-mutant EC and wild-type EC to the aberrant venous lumen expansion, we used a xenograft murine model of VM generated with a combination of TIE2-mutant EC and wild-type EC. To perform longitudinal studies, we employed a three-dimensional (3D) fibrin gel lumen formation assay and a migration assay, both using wild-type EC in competition or confrontation with TIE2-mutant EC. To investigate the mechanisms implicated in VM lumen expansion we used RNA-sequencing and short interference (sh)RNA in the TIE2-mutant EC. ResultsWe demonstrate here that in the VM xenograft model, the aberrant blood vessels were lined almost exclusively by TIE2-mutant EC, and wild-type EC were rarely found. Functionally, the TIE2-mutant EC exerted a competitive advantage over wild-type EC by inhibiting wild-type EC sprouting. In line with these findings, TIE2-mutant EC promoted repulsion of wild-type EC. ShRNA-mediated silencing of Sema3A or Sema3F in TIE2-mutant EC rescued this chemorepellent phenotype and restored the ability of wild-type EC to migrate, sprout and form lumens. Furthermore, knock-down of Sema3A or 3F in TIE2-mutant EC normalized the blood vessel size in vivo. ConclusionsOur results demonstrate that wild-type EC are not recruited to the aberrant veins suggesting VM pathogenesis is fueled by clonal expansion of TIE2-mutant EC. Mechanistically, we show that Sema3A and 3F are overexpressed in TIE2-mutant EC and play a crucial role in the pathological vascular lumen expansion in VM. Abstract (not formatted)Venous malformations (VM) are developmental defects of the vasculature characterized by tremendously enlarged and dysfunctional veins. Gain-of function somatic mutations in the endothelial-specific tyrosine kinase receptor TIE2 have been identified as one of the leading drivers of VM pathogenesis. The aim of this study was to determine whether the aberrant venous lumen expansion is caused by the recruitment of wild-type endothelial cells (EC) to the lesion or instead by TIE2-mutant EC clonal expansion. In a xenograft murine model of VM generated with a combination of TIE2-mutant EC and wild-type EC, we demonstrated that the aberrant blood vessels were lined almost exclusively by TIE2-mutant EC, suggesting lesions form by clonal expansion, while wild-type EC were not recruited to the aberrant veins. Functionally, in a three-dimensional cell competition assay, we showed that TIE2-mutant EC exerted a competitive advantage over wild-type EC by inhibiting wild-type EC sprouting and ability to form vascular lumens. In line with these findings, TIE2-mutant EC repelled wild-type EC by reversing their migration direction in a cell confrontation assay. In seeking to define the mechanism driving this repulsion phenotype, we detected elevated levels of the chemorepellent Semaphorin 3A (Sema3A) and Sema3F in the TIE2-mutant EC. ShRNA-mediated silencing of Sema3A or Sema3F rescued the chemorepellent phenotype and restored the ability of wild-type EC to migrate, sprout and form lumens. Furthermore, Sema3A or 3F knock-down in TIE2-mutant EC normalized the blood vessel morphology and size in vivo. Taken together, these data strongly indicates that Sema3A and 3F are important players in VM pathogenesis.

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

Management of recurrent gastrointestinal (GI) bleeding is a clinical unmet need for patients with Von Willebrand disease (VWD) and is linked to the presence of gut vascular malformations (angiodysplasia). We previously demonstrated that von Willebrand factor (VWF) regulates angiogenesis and vascular integrity, the likely mechanism underlying angiodysplasia. VWF controls the storage of the angiogenesis regulator Angiopoietin-2 (Angpt-2) in endothelial cells (EC), suggesting a candidate for the genesis of angiodysplasia; however, no direct evidence of the role of Angpt-2 in VWF-dependent angiogenesis is available. Here we use VWF-deficient HUVEC, and endothelial colony forming cells (ECFCs) from severe VWD patients and find that loss of VWF in EC results in increased Angpt-2 expression through a positive feedback loop via the Angpt-2-TIE2-AKT-FOXO1 pathway. We also show an imbalance of the Angpt/Tie2 pathway in vivo. In the gut of VWF-deficient mice, Angpt-2 expression is increased whilst Angpt-1 expression is decreased; this correlates with reduced expression of the pericyte marker NG2. These data suggest that VWF regulates the Angpt/Tie2 balance in the gut. To investigate the functional defects caused by loss of VWF, we use a fibrin bead assay and show that VWF-deficient HUVEC present increased sprouting. We develop a microfluidic model of 3D vasculogenesis/angiogenesis and find that ECFCs from VWD patients exhibit defective remodeling and abnormal lumen formation compared to healthy controls. Importantly, inhibition of Angpt-2 reduces sprouting in VWF-deficient HUVEC and normalises vascular networks in ECFCs from severe VWD, suggesting Angpt-2 inhibitors may be effective in VWD patients with GI bleeding and angiodysplasia. Key pointsO_LIEndothelial VWF regulates multiple steps of angiogenesis, including sprouting and lumen formation. C_LIO_LIVWF regulates Angpt-2 storage and expression, and Angpt-2 blockade normalises defective angiogenesis in VWD ECFCs. C_LI Visual Abstract O_FIG O_LINKSMALLFIG WIDTH=170 HEIGHT=200 SRC="FIGDIR/small/675051v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@150eb15org.highwire.dtl.DTLVardef@179e6beorg.highwire.dtl.DTLVardef@1bcadborg.highwire.dtl.DTLVardef@a30fb8_HPS_FORMAT_FIGEXP M_FIG VWF deficiency in HUVECs results in increased Angpt-2 release and expression via the Tie2-Akt-FOXO1 pathway. A. Model pathway for the regulation of Angpt-2 levels by VWF. Loss of VWF results in increased Angpt-2 release from endothelial cells; Angpt-2 binds to and inhibits the Tie2 receptor, decreasing its phosphorylation as well as the downstream phosphorylation of Akt and FOXO1; this leads to FOXO1 activation and an increase expression of Angpt-2, generating a feedback loop. B. Model for the pathogenesis of angiodysplasia in VWD. In the absence of VWF, the increase in Angpt-2 disrupts vascular morphogenesis through multiple mechanisms: increased sprouting, impaired vascular remodelling and an imbalance between Angpt-1 and Angpt-2 in the gut. Figure generated with Biorender. C_FIG

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.

ABSTRACT/SUMMARYBlood flow provides critical inputs for mechanisms governing vascular homeostasis. Altered hemodynamics can therefore trigger a wide range of cellular responses in blood vessels. Endothelial cells (ECs) downstream of atherosclerotic plaques for instance are exposed to turbulent flow, activating inflammatory pathways that promote immune cell infiltration. In conditions like stroke and myocardial infarction, the abrupt loss of blood flow prompts responses in vascular cells such as ECs and pericytes (PCs) to adapt to ischemic or no-flow conditions. To better understand how cerebral capillary ECs and PCs react to the sudden loss of blood flow, we used a murine brain slice model cultured for 12- and 24-hours in artificial cerebrospinal fluid (aCSF) with 95% oxygen supplementation. As expected, inflammation mediators were upregulated in cultured slices compared to non-cultured samples, particularly those associated with leukocyte recruitment. Additionally, transcriptional markers of extracellular matrix (ECM) remodeling and cell-ECM interactions were elevated, consistent with reduced PC coverage along capillaries. We initially presumed these changes reflected blood-brain barrier (BBB) degradation, but instead we found an increase in mRNA transcripts for EC junctions and stable protein levels for junction molecules, with an apparent rearrangement of Claudin5-based tight junctions. Some capillaries also exhibited reduced diameters, suggesting constriction by PCs or a subset thereof. Consistent with these observations, we found an upregulation of the vasoconstrictor Endothelin-1 (ET-1) with its receptors and contractile proteins found in a subpopulation of PCs. Suppressing ET-1 activity prevented Claudin5 upregulation, indicating that ET-1 might regulate microvascular constriction and associated changes in endothelial tight junctions. Overall, these results suggest that in the absence of blood flow, PCs contribute to capillary wall remodeling by (i) potentially mediating a mechanism driven by ET-1 that affects EC Claudin5 dynamics, and (ii) reducing capillary ECM and detaching from microvessel walls.

Thoracic aortic aneurysm is a life-threatening condition due to either genetic syndromes (e.g., Marfan syndrome) or cardiovascular risk factors (e.g., hypertension, aging and smoking), which favour the onset of sporadic thoracic aneurysms. Activation of the transforming growth factor-{beta} pathway and dysregulation of mechanotransduction signals in vascular smooth muscle cells play a key role in the development of both syndromic and sporadic forms of thoracic aortic aneurysm. The precise molecular mechanisms underlying thoracic aortic aneurysm onset and progression are still unresolved and available therapies merely rely on surgical intervention. Integrins containing the V subunit are central to both transforming growth factor-{beta} (TGF-{beta}) and mechanotransduction signalling pathways, leading to pro-fibrotic molecular events. Here we investigate the role of V integrins in the development of both syndromic and sporadic thoracic aortic aneurysms and the therapeutic potential of two V integrin inhibitors (Cilengitide and GLPG0187). We observed that V integrins are more expressed in both types of human thoracic aortic aneurysms and that integrin inhibition limits TGF-{beta} activation and mechanotransduction-related pro-fibrotic pathways in patient-derived vascular smooth muscle cells. In vivo experiments revealed that Cilengitide is the most effective V integrin inhibitor in limiting the dilation of the aortic bulb in murine models of both syndromic and sporadic forms of thoracic aortic aneurysms. These findings set the V integrin inhibitor Cilengitide as a promising drug for the treatment of thoracic aortic aneurysms.

To generate new vessels, endothelial cells (ECs) form invadosomes, which are actin-based microdomains with a proteolytic activity that degrade the basement membrane. We previously demonstrated that ECs form linear invadosomes in fibrillar type I collagen context. In this study, we aim to investigate the molecular mechanisms by which ECs guides angiogenesis in a fibrillar type I collagen context. We found that Discoidin Domain Receptor 2 (DDR2) is the collagen receptor tyrosine kinase required to form linear invadosomes in ECs. We further demonstrated that it acts in synergy with VEGF to promote extracellular matrix degradation. We highlighted the involvement of an interaction between DDR2 and the matrix metalloproteinase MMP14 in this process. Finally, using in vitro and ex-vivo angiogenesis assays, we demonstrated a pro-angiogenic function of DDR2 in a collagen-rich microenvironment. This study allows us to propose DDR2-dependent linear invadosomes as targets to modulate angiogenesis.

The extracellular matrix (ECM) supports blood vessel architecture and functionality and undergoes active remodelling during vascular repair and atherogenesis. Vascular smooth muscle cells (VSMCs) are essential for vessel repair and, via their secretome, can invade from the vessel media into the intima to mediate ECM remodelling. Accumulation of fibronectin (FN) is a hallmark of early vascular repair and atherosclerosis. Here we show that FN stimulates VSMCs to secrete small extracellular vesicles (sEVs) by activating the {beta}1 integrin/FAK/Src pathway as well as Arp2/3-dependent branching of the actin cytoskeleton. We found that sEVs are trapped by the ECM in vitro and colocalise with FN in symptomatic atherosclerotic plaques in vivo. Functionally, ECM-trapped sEVs induced the formation of focal adhesions (FA) with enhanced pulling forces at the cellular periphery preventing cellular spreading and adhesion. Proteomic and GO pathway analysis revealed that VSMC-derived sEVs display a cell adhesion signature and are specifically enriched with collagen VI on the sEV surface. In vitro assays identified collagen VI as playing a key role in cell adhesion and invasion directionality. Taken together our data suggests that the accumulation of FN is a key early event in vessel repair acting to promote secretion of collage VI enriched sEVs by VSMCs. These sEVs stimulate directional invasion, most likely by triggering peripheral focal adhesion formation and actomyosin contraction to exert sufficient traction force to enable VSMC movement within the complex vascular ECM network. Figure AbstractVascular smooth muscle cells sense fibronectin via {beta}1 integrin and secrete small extracellular vesicles loaded with collagen VI. These extracellular vesicles are entrapped in the extracellular matrix and induce formation of peripheral focal adhesions presenting adhesion complex ECM proteins including collagen VI, LGALS3BP, EDIL3 and TGFBI. Focal adhesions anchor the extracellular matrix to actin fibrils in the cell. Contraction of the actin fibrils generates the mechanical force for directional cell invasion through the matrix. This figure was created with BioRender (https://biorender.com/). O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/551257v3_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@1355004org.highwire.dtl.DTLVardef@1186e9forg.highwire.dtl.DTLVardef@106ad30org.highwire.dtl.DTLVardef@1543518_HPS_FORMAT_FIGEXP M_FIG C_FIG

Hereditary hemorrhagic telangiectasia (HHT) is a genetic vascular disorder that causes systemic arteriovenous malformations (AVMs) associated with severe complications. Angiopoietin (ANG)-2 has been identified as a consistently upregulated secreted protein across various HHT models, and neutralizing ANG2 reduces AVMs in mice. ANG2 has thus emerged as a potential target for HHT treatment. Here, we report the development of a peptide vaccine (ANG2-P3:CRM197) that selectively targets ANG2 over ANG1 and tested its effectiveness in decreasing retinal AVMs in neonatal mice injected with BMP9/10 blocking antibodies, a model of HHT. Litter groups from female C57BL/6 mice immunized with ANG2-P3:CRM197 received injections of anti-BMP9/10 antibodies, and their retinas were examined for vascular pathology. The potential toxicity of the vaccine was evaluated in females 12 months post-immunization through echocardiography, basic metabolic panels, and lipid profiles. Circulating anti-ANG2 antibodies were detected in nursing neonates of vaccinated females, with antibody levels comparable between litters and their dams, indicating effective antibody transfer from the dams. A significant decrease in AVM number and size was observed in the retinas of pups exposed to ANG2-P3:CRM197 antibodies compared to unexposed pups. Arterial and venous diameters were normalized in the vaccinated pups retinas. The vaccinated females showed no abnormalities in cardiac, liver, or kidney functions. A vaccine strategy targeting ANG2 appears safe and improves AVM pathology in HHT mice. These findings further support the potential of inhibiting ANG2 as a viable approach for treating AVMs in HHT. KEY POINTSO_LIA peptide vaccine that targets ANG2 reduced AVM number and size, and normalized arterial and venous diameters in a mouse model of HHT. C_LIO_LIThe vaccine did not cause any abnormalities in cardiac, liver, or kidney functions, and thus appeared safe. C_LI

Dysfunctional lymphangiogenesis is a component of several diseases with hypoxic microenvironments, including secondary lymphedema and solid malignancies. These vessels are ineffective at draining interstitial fluid, resulting in complications such as increased inflammation, slowed wound healing, and, for cancer patients, increased risk of metastasis. Current treatments to normalize vasculature have negative effects on healthy vessels and do not specifically target lymphatic endothelial cells (LECs). As hypoxia is known to change endothelial cell metabolism, exploiting LEC-specific metabolic pathways may provide a focused approach to restoring lymphatic function in patients. However, outside of glycolysis, changes to LEC metabolism in hypoxic conditions are understudied. To address this gap in knowledge, we examined the impact of glutamine availability on factors critical to lymphangiogenesis, including glycolysis, cell proliferation, and migration. We found that increasing glutamine availability results in increased lactate production as well as a hypoxia-specific increase in glycolytic genes HK2, GLUT1, and GLUT3. The presence of glutamine also encouraged LEC proliferation, while blocking glutamine transport reduced lactate production, HK2 expression, and slowed collective LEC migration. In a vessel formation assay, we found that glutamine increased vessel formation in normoxic conditions, but lowered vessel connectivity in hypoxic conditions, reflecting the dysfunction seen in hypoxic diseases. However, attenuating glycolysis by blocking glutamine transport caused LECs to form longer, interconnected vascular networks. This study reveals that glutamine availability can modulate LEC glycolysis, and therefore lymphangiogenesis, in a hypoxia-dependent manner. Collectively, our study identifies glutamine availability as a potential target for lymphatic vessel normalization in chronic and hypoxic diseases.

BackgroundHeparan sulfates (HS) attached to the apical surface of vascular endothelial cells (ECs) play an important role in regulating endothelial permeability and ligand recognition by cell-surface receptors. Shedding of heparan sulfate (HS) from the EC surface increases vascular leakage and is associated with vascular diseases. Recently, heparanase 2 (Hpa2) was described as a novel regulatory molecule that controls HS shedding. However, its role in regulating HS physiology in the vascular endothelium is largely unknown. Here, we characterize the role of endogenous Hpa2 in the vertebrate vascular system. MethodsWe use zebrafish larvae as our primary animal model. Hpa2 expression and localization was examined by in situ hybridization and immunofluorescence. Hpa2 loss-of-function (LOF) was induced by CRISPR-Cas9 or morpholino antisense strategies. We assessed vascular permeability, blood vessel architecture, and EC morphology using transgenic zebrafish and transmission electron microscopy. EC expression profiles and HS quantity were analyzed in Hpa2-LOF larvae. The capacity of recombinant Hpa2 to modulate signaling in ECs by the HS-binding growth factors fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor A165 (VEGFA165) was tested by western blotting and immunofluorescence. Attenuation of the Hpa2-LOF phenotype was tested in vivo in zebrafish larvae via use of recombinant Hpa2 and pharmacological inhibition of FGF and VEGFA signaling. ResultsWe detected hpse2 expression in hepatic tissue and localized the protein in blood vessels. Hpa2-LOF larvae exhibited increased vascular permeability, occasional hypersprouting, and altered EC and extracellular matrix (ECM) morphology. Hpa2-LOF also reduced HS levels and caused changes in the endothelial transcriptome characterized by dysregulated genes involved in ECM-receptor interaction and signal transduction regulation. Recombinant hHpa2 rescued the Hpa2-LOF phenotype in zebrafish. We showed in vitro that Hpa2 competes with FGF2 and VEGFA165 for binding on the EC surface and consequently reduces the cellular response these factors elicit. Pharmacological inhibition of these pathways alleviated the Hpa2-LOF phenotype in zebrafish. ConclusionWe conclude that Hpa2 is a circulating molecule that maintains vascular integrity by regulating HS-dependent processes on the EC surface. These results may translate into novel strategies applying recombinant Hpa2 to treat microvascular diseases.

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.

Post brain colonization, cancer cells may adhere to and spread along the abluminal surface of the vasculature providing advantageous access to oxygen, nutrients, and vessel-derived paracrine factors. For example, brain-tropic melanoma cells are well-known to invade along or within blood vessels at the invasive front, but the molecular mechanisms that guide this process are not well-characterized. We have used melanoma cells of different phenotypic states (melanocytic versus mesenchymal) to characterize different modes of perivascular invasion in the brain. We find that Sox9hi mesenchymal state melanoma cells undergo pericyte-like spreading along brain blood vessels via a Snail1-dependent process; in contrast, Sox10hi melanocytic state melanoma form proliferative, perivascular clusters. Snai1 deletion in mesenchymal-state melanoma cells diminishes Tgf{beta}-induced expression of Pdgfr{beta} which impairs Tgf{beta}/Pdgf ligand-driven motility along the brain microvasculature and dramatically reduces perivascular dispersal of melanoma cells throughout the brain post-colonization. These data suggest that, depending on their transcriptional/phenotypic state, some melanoma cells may appropriate signals that typically mediate endothelial cell:pericyte cross talk as an adaptive mechanism for maintaining vessel proximity and motility within the brain microenvironment.

BackgroundHereditary Hemorrhagic Telangiectasia type 2 (HHT2) is a genetic disorder caused by mutations in the ALK1 (ACVRL1) gene, encoding a receptor for Bone Morphogenetic Proteins 9 and 10 (BMP9/BMP10). HHT2 patients frequently develop brain arteriovenous malformations (bAVMs), which are abnormal connections between arteries and veins. Currently, surgical resection is the only treatment, associated with significant risks and complications. Despite evidence suggesting endothelial cell (EC) heterogeneity in bAVMs, it remains poorly characterized, limiting our ability to identify new therapeutic avenues. MethodsWe employed endothelial cell-specific and inducible Alk1 knockout mice (Alk1iECKO) with tamoxifen-induced deletion at postnatal day 6 (P6). We separately analyzed the P8 perineural (PNVP) and intraneural (INVP) vascular plexuses, which differ in vessel composition and flow dynamics. Single-cell RNA sequencing (scRNAseq) was performed to characterize EC heterogeneity and identify transcriptomic changes in both vascular plexuses of mutant versus wild type mice. ResultsLoss of endothelial ALK1 signaling triggered bAVM formation predominantly in the PNVP vascular network. scRNAseq revealed that Alk1 deletion promoted brain capillaries differentiation into angiogenic-1 ECs, whereas it drove PNVP venules EC proliferation and the emergence of the unique angiogenic-2 cluster. The latter shares transcriptomic features with human AVM ECs, including angiogenic tip cell markers and a strong glycolytic signature. Among its defining markers, Kit emerged as a direct downstream target of BMP9-ALK1 signaling. Pharmacological KIT inhibition using Masitinib, Imatinib, or KIT-blocking antibodies prevented bAVM formation in Alk1iECKO mice. ConclusionOur study uncovers a previously unrecognized EC population, the angiogenic-2 cluster, as a key contributor to bAVM development. We identify Kit as a central regulator of this cluster, establishing it as a promising therapeutic target for preventing bAVMs in HHT2. Clinical PerspectiveO_ST_ABSWhat is new?C_ST_ABSO_LIUsing endothelial-specific Alk1 knockout mouse models and single-cell transcriptomics, we identified a novel angiogenic endothelial cell population as a key driver of brain AVM formation. C_LIO_LIThis angiogenic EC cluster shares molecular features with human bAVM cells, including high expression of KIT, which we identified as a new direct transcriptional target of BMP9-ALK1 signaling. C_LIO_LIPharmacological inhibition of KIT using small molecules or blocking antibodies effectively prevents AVM formation in vivo, establishing KIT as a promising therapeutic target. C_LI What are the clinical implications?O_LIOur findings support the therapeutic potential of targeting KIT with FDA-approved drugs such as Imatinib to treat HHT2-associated brain AVMs, offering a non-invasive alternative to surgical intervention. C_LIO_LICharacterizing AVM-specific endothelial subtypes may enable the development of targeted and personalized therapies, improving patient outcomes and minimizing treatment-associated risks in HHT. C_LI