Role of endothelial PDGFB in arterio-venous malformations pathogenesis.

Arterial-venous malformations (AVMs) are direct connections between arteries and veins without an intervening capillary bed. Either familial inherited or sporadically occurring, localized pericytes (PCs) drop is among the AVMs' hallmarks. Whether impaired PC coverage triggers AVMs or it is a secondary event is unclear. Here we evaluated the role of the master regulator of PC recruitment, Platelet derived growth factor B (PDGFB) in AVM pathogenesis. Using tamoxifen-inducible deletion of Pdgfb in endothelial cells (ECs), we show that disruption of EC Pdgfb-mediated PC recruitment and maintenance leads to capillary enlargement and organotypic AVM-like structures. These vascular lesions contain non-proliferative hyperplastic, hypertrophic and miss-oriented capillary ECs with an altered capillary EC fate identity. Mechanistically, we propose that PDGFB maintains capillary EC size and caliber to limit hemodynamic changes, thus restricting expression of Krüppel like factor 4 and activation of Bone morphogenic protein, Transforming growth factor ß and NOTCH signaling in ECs. Furthermore, our study emphasizes that inducing or activating PDGFB signaling may be a viable therapeutic approach for treating vascular malformations.

ALK1 controls hepatic vessel formation, angiodiversity, and angiocrine functions in hereditary hemorrhagic telangiectasia of the liver.

BACKGROUND AND AIMS: In hereditary hemorrhagic telangiectasia (HHT), severe liver vascular malformations are associated with mutations in the Activin A Receptor-Like Type 1 ( ACVRL1 ) gene encoding ALK1, the receptor for bone morphogenetic protein (BMP) 9/BMP10, which regulates blood vessel development. Here, we established an HHT mouse model with exclusive liver involvement and adequate life expectancy to investigate ALK1 signaling in liver vessel formation and metabolic function. APPROACH AND RESULTS: Liver sinusoidal endothelial cell (LSEC)-selective Cre deleter line, Stab2-iCreF3 , was crossed with Acvrl1 -floxed mice to generate LSEC-specific Acvrl1 -deficient mice ( Alk1HEC-KO ). Alk1HEC-KO mice revealed hepatic vascular malformations and increased posthepatic flow, causing right ventricular volume overload. Transcriptomic analyses demonstrated induction of proangiogenic/tip cell gene sets and arterialization of hepatic vessels at the expense of LSEC and central venous identities. Loss of LSEC angiokines Wnt2 , Wnt9b , and R-spondin-3 ( Rspo3 ) led to disruption of metabolic liver zonation in Alk1HEC-KO mice and in liver specimens of patients with HHT. Furthermore, prion-like protein doppel ( Prnd ) and placental growth factor ( Pgf ) were upregulated in Alk1HEC-KO hepatic endothelial cells, representing candidates driving the organ-specific pathogenesis of HHT. In LSEC in vitro , stimulation or inhibition of ALK1 signaling counter-regulated Inhibitors of DNA binding (ID)1-3, known Alk1 transcriptional targets. Stimulation of ALK1 signaling and inhibition of ID1-3 function confirmed regulation of Wnt2 and Rspo3 by the BMP9/ALK1/ID axis. CONCLUSIONS: Hepatic endothelial ALK1 signaling protects from development of vascular malformations preserving organ-specific endothelial differentiation and angiocrine signaling. The long-term surviving Alk1HEC-KO HHT model offers opportunities to develop targeted therapies for this severe disease.

RNA-Binding Proteins Regulate Post-Transcriptional Responses to TGF-ß to Coordinate Function and Mesenchymal Activation of Murine Endothelial Cells.

BACKGROUND: Endothelial cells (ECs) are primed to respond to various signaling cues. For example, TGF (transforming growth factor)-ß has major effects on EC function and phenotype by driving ECs towards a more mesenchymal state (ie, triggering endothelial to mesenchymal activation), a dynamic process associated with cardiovascular diseases. Although transcriptional regulation triggered by TGF-ß in ECs is well characterized, post-transcriptional regulatory mechanisms induced by TGF-ß remain largely unknown. METHODS: Using RNA interactome capture, we identified global TGF-ß driven changes in RNA-binding proteins in ECs. We investigated specific changes in the RNA-binding patterns of hnRNP H1 (heterogeneous nuclear ribonucleoprotein H1) and Csde1 (cold shock domain containing E1) using RNA immunoprecipitation and overlapped this with RNA-sequencing data after knockdown of either protein for functional insight. Using a modified proximity ligation assay, we visualized the specific interactions between hnRNP H1 and Csde1 and target RNAs in situ both in vitro and in mouse heart sections. RESULTS: Characterization of TGF-ß-regulated RBPs (RNA-binding proteins) revealed hnRNP H1 and Csde1 as key regulators of the cellular response to TGF-ß at the post-transcriptional level, with loss of either protein-promoting mesenchymal activation in ECs. We found that TGF-ß drives an increase in binding of hnRNP H1 to its target RNAs, offsetting mesenchymal activation, but a decrease in Csde1 RNA-binding, facilitating this process. Both, hnRNP H1 and Csde1, dynamically bind and regulate specific subsets of mRNAs related to mesenchymal activation and endothelial function. CONCLUSIONS: Together, we show that RBPs play a key role in the endothelial response to TGF-ß stimulation at the post-transcriptional level and that the RBPs hnRNP H1 and Csde1 serve to maintain EC function and counteract mesenchymal activation. We propose that TGF-ß profoundly modifies RNA-protein interaction entailing feedback and feed-forward control at the post-transcriptional level, to fine-tune mesenchymal activation in ECs.

Executive summary of the 14th HHT international scientific conference.

Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal dominant vascular disorder characterized by small, dilated clustered vessels (telangiectasias) and by larger visceral arteriovenous malformations (AVMs), which directly connect the feeding arteries with the draining veins. These lesions are fragile, prone to rupture, and lead to recurrent epistaxis and/or internal hemorrhage among other complications. Germline heterozygous loss-of-function (LOF) mutations in Bone Morphogenic Protein 9 (BMP9) and BMP10 signaling pathway genes (endoglin-ENG, activin like kinase 1 ACVRL1 aka ALK1, and SMAD4) cause different subtypes of HHT (HHT1, HHT2 and HHT-juvenile polyposis (JP)) and have a worldwide combined incidence of about 1:5000. Expert clinicians and international scientists gathered in Cascais, Portugal from September 29th to October 2nd, 2022 to present the latest scientific research in the HHT field and novel treatment strategies for people living with HHT. During the largest HHT scientific conference yet, participants included 293 in person and 46 virtually. An impressive 209 abstracts were accepted to the meeting and 59 were selected for oral presentations. The remaining 150 abstracts were presented during judged poster sessions. This review article summarizes the basic and clinical abstracts selected as oral presentations with their new observations and discoveries as well as surrounding discussion and debate. Two discussion-based workshops were also held during the conference, each focusing on mechanisms and clinical perspectives in either AVM formation and progression or current and future therapies for HHT. Our hope is that this paper will represent the current progress and the remaining unanswered questions surrounding HHT, in order to serve as an update for those within the field and an invitation to those scientists and clinicians as yet outside of the field of HHT.

SMAD4 Prevents Flow Induced Arteriovenous Malformations by Inhibiting Casein Kinase 2.

BACKGROUND: Hereditary hemorrhagic telangiectasia (HHT) is an inherited vascular disorder that causes arteriovenous malformations (AVMs). Mutations in the genes encoding Endoglin ( ENG) and activin-receptor-like kinase 1 ( AVCRL1 encoding ALK1) cause HHT type 1 and 2, respectively. Mutations in the SMAD4 gene are present in families with juvenile polyposis-HHT syndrome that involves AVMs. SMAD4 is a downstream effector of transforming growth factor-ß (TGFß)/bone morphogenetic protein (BMP) family ligands that signal via activin-like kinase receptors (ALKs). Ligand-neutralizing antibodies or inducible, endothelial-specific Alk1 deletion induce AVMs in mouse models as a result of increased PI3K (phosphatidylinositol 3-kinase)/AKT (protein kinase B) signaling. Here we addressed if SMAD4 was required for BMP9-ALK1 effects on PI3K/AKT pathway activation. METHODS: The authors generated tamoxifen-inducible, postnatal, endothelial-specific Smad4 mutant mice ( Smad4iΔEC). RESULTS: We found that loss of endothelial Smad4 resulted in AVM formation and lethality. AVMs formed in regions with high blood flow in developing retinas and other tissues. Mechanistically, BMP9 signaling antagonized flow-induced AKT activation in an ALK1- and SMAD4-dependent manner. Smad4iΔEC endothelial cells in AVMs displayed increased PI3K/AKT signaling, and pharmacological PI3K inhibitors or endothelial Akt1 deletion both rescued AVM formation in Smad4iΔEC mice. BMP9-induced SMAD4 inhibited casein kinase 2 ( CK2) transcription, in turn limiting PTEN phosphorylation and AKT activation. Consequently, CK2 inhibition prevented AVM formation in Smad4iΔEC mice. CONCLUSIONS: Our study reveals SMAD4 as an essential effector of BMP9-10/ALK1 signaling that affects AVM pathogenesis via regulation of CK2 expression and PI3K/AKT1 activation.

Correction: GDNF Overexpression from the Native Locus Reveals its Role in the Nigrostriatal Dopaminergic System Function.

[This corrects the article DOI: 10.1371/journal.pgen.1005710.].

Alk2/ACVR1 and Alk3/BMPR1A Provide Essential Function for Bone Morphogenetic Protein-Induced Retinal Angiogenesis.

OBJECTIVE: Increasing evidence suggests that bone morphogenetic protein (BMP) signaling regulates angiogenesis. Here, we aimed to define the function of BMP receptors in regulating early postnatal angiogenesis by analysis of inducible, endothelial-specific deletion of the BMP receptor components Bmpr2 (BMP type 2 receptor), Alk1 (activin receptor-like kinase 1), Alk2, and Alk3 in mouse retinal vessels. APPROACH AND RESULTS: Expression analysis of several BMP ligands showed that proangiogenic BMP ligands are highly expressed in postnatal retinas. Consistently, BMP receptors are also strongly expressed in retina with a distinct pattern. To assess the function of BMP signaling in retinal angiogenesis, we first generated mice carrying an endothelial-specific inducible deletion of Bmpr2. Postnatal deletion of Bmpr2 in endothelial cells substantially decreased the number of angiogenic sprouts at the vascular front and branch points behind the front, leading to attenuated radial expansion. To identify critical BMPR1s (BMP type 1 receptors) associated with BMPR2 in retinal angiogenesis, we generated endothelial-specific inducible deletion of 3 BMPR1s abundantly expressed in endothelial cells and analyzed the respective phenotypes. Among these, endothelial-specific deletion of either Alk2/acvr1 or Alk3/Bmpr1a caused a delay in radial expansion, reminiscent of vascular defects associated with postnatal endothelial-specific deletion of BMPR2, suggesting that ALK2/ACVR1 and ALK3/BMPR1A are likely to be the critical BMPR1s necessary for proangiogenic BMP signaling in retinal vessels. CONCLUSIONS: Our data identify BMP signaling mediated by coordination of ALK2/ACVR1, ALK3/BMPR1A, and BMPR2 as an essential proangiogenic cue for retinal vessels.

GDNF Overexpression from the Native Locus Reveals its Role in the Nigrostriatal Dopaminergic System Function.

Degeneration of nigrostriatal dopaminergic system is the principal lesion in Parkinson's disease. Because glial cell line-derived neurotrophic factor (GDNF) promotes survival of dopamine neurons in vitro and in vivo, intracranial delivery of GDNF has been attempted for Parkinson's disease treatment but with variable success. For improving GDNF-based therapies, knowledge on physiological role of endogenous GDNF at the sites of its expression is important. However, due to limitations of existing genetic model systems, such knowledge is scarce. Here, we report that prevention of transcription of Gdnf 3'UTR in Gdnf endogenous locus yields GDNF hypermorphic mice with increased, but spatially unchanged GDNF expression, enabling analysis of postnatal GDNF function. We found that increased level of GDNF in the central nervous system increases the number of adult dopamine neurons in the substantia nigra pars compacta and the number of dopaminergic terminals in the dorsal striatum. At the functional level, GDNF levels increased striatal tissue dopamine levels and augmented striatal dopamine release and re-uptake. In a proteasome inhibitor lactacystin-induced model of Parkinson's disease GDNF hypermorphic mice were protected from the reduction in striatal dopamine and failure of dopaminergic system function. Importantly, adverse phenotypic effects associated with spatially unregulated GDNF applications were not observed. Enhanced GDNF levels up-regulated striatal dopamine transporter activity by at least five fold resulting in enhanced susceptibility to 6-OHDA, a toxin transported into dopamine neurons by DAT. Further, we report how GDNF levels regulate kidney development and identify microRNAs miR-9, miR-96, miR-133, and miR-146a as negative regulators of GDNF expression via interaction with Gdnf 3'UTR in vitro. Our results reveal the role of GDNF in nigrostriatal dopamine system postnatal development and adult function, and highlight the importance of correct spatial expression of GDNF. Furthermore, our results suggest that 3'UTR targeting may constitute a useful tool in analyzing gene function.

Targeting NCK-Mediated Endothelial Cell Front-Rear Polarity Inhibits Neovascularization.

BACKGROUND: Sprouting angiogenesis is a key process driving blood vessel growth in ischemic tissues and an important drug target in a number of diseases, including wet macular degeneration and wound healing. Endothelial cells forming the sprout must develop front-rear polarity to allow sprout extension. The adaptor proteins Nck1 and 2 are known regulators of cytoskeletal dynamics and polarity, but their function in angiogenesis is poorly understood. Here, we show that the Nck adaptors are required for endothelial cell front-rear polarity and migration downstream of the angiogenic growth factors VEGF-A and Slit2. METHODS AND RESULTS: Mice carrying inducible, endothelial-specific Nck1/2 deletions fail to develop front-rear polarized vessel sprouts and exhibit severe angiogenesis defects in the postnatal retina and during embryonic development. Inactivation of NCK1 and 2 inhibits polarity by preventing Cdc42 and Pak2 activation by VEGF-A and Slit2. Mechanistically, NCK binding to ROBO1 is required for both Slit2- and VEGF-induced front-rear polarity. Selective inhibition of polarized endothelial cell migration by targeting Nck1/2 prevents hypersprouting induced by Notch or Bmp signaling inhibition, and pathological ocular neovascularization and wound healing, as well. CONCLUSIONS: These data reveal a novel signal integration mechanism involving NCK1/2, ROBO1/2, and VEGFR2 that controls endothelial cell front-rear polarity during sprouting angiogenesis.

Shear stress and pathophysiological PI3K involvement in vascular malformations.

Molecular characterization of vascular anomalies has revealed that affected endothelial cells (ECs) harbor gain-of-function (GOF) mutations in the gene encoding the catalytic α subunit of PI3Kα (PIK3CA). These PIK3CA mutations are known to cause solid cancers when occurring in other tissues. PIK3CA-related vascular anomalies, or "PIKopathies," range from simple, i.e., restricted to a particular form of malformation, to complex, i.e., presenting with a range of hyperplasia phenotypes, including the PIK3CA-related overgrowth spectrum. Interestingly, development of PIKopathies is affected by fluid shear stress (FSS), a physiological stimulus caused by blood or lymph flow. These findings implicate PI3K in mediating physiological EC responses to FSS conditions characteristic of lymphatic and capillary vessel beds. Consistent with this hypothesis, increased PI3K signaling also contributes to cerebral cavernous malformations, a vascular disorder that affects low-perfused brain venous capillaries. Because the GOF activity of PI3K and its signaling partners are excellent drug targets, understanding PIK3CA's role in the development of vascular anomalies may inform therapeutic strategies to normalize EC responses in the diseased state. This Review focuses on PIK3CA's role in mediating EC responses to FSS and discusses current understanding of PIK3CA dysregulation in a range of vascular anomalies that particularly affect low-perfused regions of the vasculature. We also discuss recent surprising findings linking increased PI3K signaling to fast-flow arteriovenous malformations in hereditary hemorrhagic telangiectasias.

SMAD4 maintains the fluid shear stress set point to protect against arterial-venous malformations.

Vascular networks form, remodel, and mature under the influence of both fluid shear stress (FSS) and soluble factors. Physiological FSS promotes and maintains vascular stability via synergy with bone morphogenic proteins 9 and 10 (BMP9 and BMP10). Conversely, mutation of the BMP receptors activin-like kinase 1 (ALK1), endoglin (ENG), or the downstream effector, SMAD family member 4 (SMAD4) leads to hereditary hemorrhagic telangiectasia (HHT), characterized by fragile and leaky arterial-venous malformations (AVMs). How endothelial cells (ECs) integrate FSS and BMP signals in vascular development and homeostasis and how mutations give rise to vascular malformations is not well understood. Here, we aimed to elucidate the mechanism of synergy between FSS and SMAD signaling in vascular stability and how disruption of this synergy leads to AVMs. We found that loss of Smad4 increased the sensitivity of ECs to flow by lowering the FSS set point, with resulting AVMs exhibiting features of excessive flow-mediated morphological responses. Mechanistically, loss of SMAD4 disinhibits flow-mediated KLF4-TIE2-PI3K/Akt signaling, leading to cell cycle progression-mediated loss of arterial identity due to KLF4-mediated repression of cyclin dependent Kinase (CDK) inhibitors CDKN2A and CDKN2B. Thus, AVMs caused by Smad4 deletion are characterized by chronic high flow remodeling with excessive EC proliferation and loss of arterial identity as triggering events.

Inactivation of Palb2 gene leads to mesoderm differentiation defect and early embryonic lethality in mice.

Mutations of the PALB2 tumor suppressor gene in humans are associated with hereditary predisposition to breast and also some other cancers. In the present study, we have characterized mice deficient in Palb2. The data show that the Palb2((+/-)) mice are normal and fertile, and lack macroscopic tumors when followed up till the age of 8 months. Homozygous (HO) Palb2((-/-)) mice present with embryonic lethality and die at E9.5 at the latest. The mutant embryos are smaller in size, developmentally retarded and display defective mesoderm differentiation after gastrulation. In Palb2((-/-)) embryos, the expression of cyclin-dependent kinase inhibitor p21 is increased, and Palb2((-/-)) blastocysts show a growth defect in vitro. Hence, the phenotype of the Palb2((-/-)) mice in many regards resembles those previously reported for Brca1 and Brca2 knockout mice. The similarity in the phenotypes between Palb2, Brca1 and Brca2 knockout mice further supports the functional relationship shown in vitro for these three proteins. Accordingly, our data in vivo suggest that a key function for PALB2 is to interact with and to build up appropriate communication between BRCA1 and BRCA2, thereby licensing the successful performance of the physiological tasks mediated by these two proteins, particularly in homologous recombination and in proper DNA damage response signaling.

Sema4C-Plexin B2 signalling modulates ureteric branching in developing kidney.

Semaphorins, originally identified as axon guidance molecules, have also been implicated in angiogenesis, function of the immune system and cancerous growth. Here we show that deletion of Plexin B2 (Plxnb2), a semaphorin receptor that is expressed both in the pretubular aggregates and the ureteric epithelium in the developing kidney, results in renal hypoplasia and occasional double ureters. The rate of cell proliferation in the ureteric epithelium and consequently the number of ureteric tips are reduced in the kidneys lacking Plexin B2 (Plxnb2-/-). Semaphorin 4C, a ligand for Plexin B2, stimulates branching of the ureteric epithelium in wild type and Plxnb2+/- kidney explants, but not in Plxnb2-/- explants. As shown by co-immunoprecipitation Plexin B2 interacts with the Ret receptor tyrosine kinase, the receptor of Glial-cell-line-derived neurotrophic factor (Gdnf), in embryonic kidneys. Isolated Plxnb2-/- ureteric buds fail to respond to Gdnf by branching, but this response is rescued by Fibroblast growth factor 7 and Follistatin as well as by the metanephric mesenchyme. The differentiation of the nephrogenic mesenchyme, its morphology and the rate of apoptosis in the Plxnb2-/- kidneys are normal. Plexin B2 is co-expressed with Plexin B1 (Plxnb1) in the kidney. The double homozygous Plxnb1-Plxnb2-deficient mice show high embryonic lethality prior to onset of nephrogenesis. The only double homozygous embryo surviving to E12 showed hypoplastic kidneys with ureteric branches and differentiating mesenchyme. Taken together, our results show that Sema4C-Plexin B2 signalling regulates ureteric branching, possibly through modulation of Gdnf signalling by interaction with Ret, and suggest non-redundant roles for Plexin B1 and Plexin B2 in kidney development.

The GDNF target Vsnl1 marks the ureteric tip.

Glial cell line-derived neurotrophic factor (GDNF) is indispensable for ureteric budding and branching. If applied exogenously, GDNF promotes ectopic ureteric buds from the Wolffian duct. Although several downstream effectors of GDNF are known, the identification of early response genes is incomplete. Here, microarray screening detected several GDNF-regulated genes in the Wolffian duct, including Visinin like 1 (Vsnl1), which encodes a neuronal calcium-sensor protein. We observed renal Vsnl1 expression exclusively in the ureteric epithelium, but not in Gdnf-null kidneys. In the tissue culture of Gdnf-deficient kidney primordium, exogenous GDNF and alternative bud inducers (FGF7 and follistatin) restored Vsnl1 expression. Hence, Vsnl1 characterizes the tip of the ureteric bud epithelium regardless of the inducer. In the tips, Vsnl1 showed a mosaic expression pattern that was mutually exclusive with ß-catenin transcriptional activation. Vsnl1 was downregulated in both ß-catenin-stabilized and ß-catenin-deficient kidneys. Moreover, in a mouse collecting duct cell line, Vsnl1 compromised ß-catenin stability, suggesting a counteracting relationship between Vsnl1 and ß-catenin. In summary, Vsnl1 marks ureteric bud tips in embryonic kidneys, and its mosaic pattern demonstrates a heterogeneity of cell types that may be critical for normal ureteric branching.

Analysis of myocardial cellular gene expression during pressure overload reveals matrix based functional intercellular communication.

To identify cellular mechanisms responsible for pressure overload triggered heart failure, we isolated cardiomyocytes, endothelial cells, and fibroblasts as most abundant cell types from mouse hearts in the subacute and chronic stages after transverse aortic constriction (TAC) and performed RNA-sequencing. We detected highly cell-type specific transcriptional responses with characteristic time courses and active intercellular communication. Cardiomyocytes after TAC exerted an early and sustained upregulation of inflammatory and matrix genes and a concomitant suppression of metabolic and ion channel genes. Fibroblasts, in contrast, showed transient early upregulation of inflammatory and matrix genes and downregulation of angiogenesis genes, but sustained induction of cell cycle and ion channel genes during TAC. Endothelial cells transiently induced cell cycle and extracellular matrix genes early after TAC, but exerted a long-lasting upregulation of inflammatory genes. As we found that matrix production by multiple cell types triggers pathological cellular responses, it might serve as a future therapeutic target.

Development of the urogenital system is regulated via the 3'UTR of GDNF.

Mechanisms controlling ureter lenght and the position of the kidney are poorly understood. Glial cell-line derived neurotrophic factor (GDNF) induced RET signaling is critical for ureteric bud outgrowth, but the function of endogenous GDNF in further renal differentiation and urogenital system development remains discursive. Here we analyzed mice where 3' untranslated region (UTR) of GDNF is replaced with sequence less responsive to microRNA-mediated regulation, leading to increased GDNF expression specifically in cells naturally transcribing Gdnf. We demonstrate that increased Gdnf leads to short ureters in kidneys located in an abnormally caudal position thus resembling human pelvic kidneys. High GDNF levels expand collecting ductal progenitors at the expense of ureteric trunk elongation and result in expanded tip and short trunk phenotype due to changes in cell cycle length and progenitor motility. MEK-inhibition rescues these defects suggesting that MAPK-activity mediates GDNF's effects on progenitors. Moreover, Gdnf   hyper mice are infertile likely due to effects of excess GDNF on distal ureter remodeling. Our findings suggest that dysregulation of GDNF levels, for example via alterations in 3'UTR, may account for a subset of congenital anomalies of the kidney and urinary tract (CAKUT) and/or congenital infertility cases in humans and pave way to future studies.

Endophilin-A2 dependent VEGFR2 endocytosis promotes sprouting angiogenesis.

Endothelial cell migration, proliferation and survival are triggered by VEGF-A activation of VEGFR2. However, how these cell behaviors are regulated individually is still unknown. Here we identify Endophilin-A2 (ENDOA2), a BAR-domain protein that orchestrates CLATHRIN-independent internalization, as a critical mediator of endothelial cell migration and sprouting angiogenesis. We show that EndoA2 knockout mice exhibit postnatal angiogenesis defects and impaired front-rear polarization of sprouting tip cells. ENDOA2 deficiency reduces VEGFR2 internalization and inhibits downstream activation of the signaling effector PAK but not ERK, thereby affecting front-rear polarity and migration but not proliferation or survival. Mechanistically, VEGFR2 is directed towards ENDOA2-mediated endocytosis by the SLIT2-ROBO pathway via SLIT-ROBO-GAP1 bridging of ENDOA2 and ROBO1. Blocking ENDOA2-mediated endothelial cell migration attenuates pathological angiogenesis in oxygen-induced retinopathy models. This work identifies a specific endocytic pathway controlling a subset of VEGFR2 mediated responses that could be targeted to prevent excessive sprouting angiogenesis in pathological conditions.

Lacteal junction zippering protects against diet-induced obesity.

Excess dietary lipid uptake causes obesity, a major global health problem. Enterocyte-absorbed lipids are packaged into chylomicrons, which enter the bloodstream through intestinal lymphatic vessels called lacteals. Here, we show that preventing lacteal chylomicron uptake by inducible endothelial genetic deletion of Neuropilin1 (Nrp1) and Vascular endothelial growth factor receptor 1 (Vegfr1; also known as Flt1) renders mice resistant to diet-induced obesity. Absence of NRP1 and FLT1 receptors increased VEGF-A bioavailability and signaling through VEGFR2, inducing lacteal junction zippering and chylomicron malabsorption. Restoring permeable lacteal junctions by VEGFR2 and vascular endothelial (VE)-cadherin signaling inhibition rescued chylomicron transport in the mutant mice. Zippering of lacteal junctions by disassembly of cytoskeletal VE-cadherin anchors prevented chylomicron uptake in wild-type mice. These data suggest that lacteal junctions may be targets for preventing dietary fat uptake.

Defective fluid shear stress mechanotransduction mediates hereditary hemorrhagic telangiectasia.

Morphogenesis of the vascular system is strongly modulated by mechanical forces from blood flow. Hereditary hemorrhagic telangiectasia (HHT) is an inherited autosomal-dominant disease in which arteriovenous malformations and telangiectasias accumulate with age. Most cases are linked to heterozygous mutations in Alk1 or Endoglin, receptors for bone morphogenetic proteins (BMPs) 9 and 10. Evidence suggests that a second hit results in clonal expansion of endothelial cells to form lesions with poor mural cell coverage that spontaneously rupture and bleed. We now report that fluid shear stress potentiates BMPs to activate Alk1 signaling, which correlates with enhanced association of Alk1 and endoglin. Alk1 is required for BMP9 and flow responses, whereas endoglin is only required for enhancement by flow. This pathway mediates both inhibition of endothelial proliferation and recruitment of mural cells; thus, its loss blocks flow-induced vascular stabilization. Identification of Alk1 signaling as a convergence point for flow and soluble ligands provides a molecular mechanism for development of HHT lesions.

PI3 kinase inhibition improves vascular malformations in mouse models of hereditary haemorrhagic telangiectasia.

Activin receptor-like kinase 1 (ALK1) is an endothelial serine-threonine kinase receptor for bone morphogenetic proteins (BMPs) 9 and 10. Inactivating mutations in the ALK1 gene cause hereditary haemorrhagic telangiectasia type 2 (HHT2), a disabling disease characterized by excessive angiogenesis with arteriovenous malformations (AVMs). Here we show that inducible, endothelial-specific homozygous Alk1 inactivation and BMP9/10 ligand blockade both lead to AVM formation in postnatal retinal vessels and internal organs including the gastrointestinal (GI) tract in mice. VEGF and PI3K/AKT signalling are increased on Alk1 deletion and BMP9/10 ligand blockade. Genetic deletion of the signal-transducing Vegfr2 receptor prevents excessive angiogenesis but does not fully revert AVM formation. In contrast, pharmacological PI3K inhibition efficiently prevents AVM formation and reverts established AVMs. Thus, Alk1 deletion leads to increased endothelial PI3K pathway activation that may be a novel target for the treatment of vascular lesions in HHT2.