Heart Failure Strategically Focused Research Network: Summary of Results and Future Directions.

Heart failure remains among the most common and morbid health conditions. The Heart Failure Strategically Focused Research Network (HF SFRN) was funded by the American Heart Association to facilitate collaborative, high-impact research in the field of heart failure across the domains of basic, clinical, and population research. The Network was also charged with developing training opportunities for young investigators. Four centers were funded in 2016: Duke University, University of Colorado, University of Utah, and Massachusetts General Hospital-University of Massachusetts. This report summarizes the aims of each center and major research accomplishments, as well as training outcomes from the HF SFRN.

Tumor-specific interendothelial adhesion mediated by FLRT2 facilitates cancer aggressiveness.

Blood vessel abnormalization alters cancer cell metabolism and promotes cancer dissemination and metastasis. However, the biological features of the abnormalized blood vessels that facilitate cancer progression and whether they can be targeted therapeutically have not been fully investigated. Here, we found that an axon guidance molecule, fibronectin leucine-rich transmembrane protein 2 (FLRT2), is expressed preferentially in abnormalized vessels of advanced colorectal cancers in humans and that its expression correlates negatively with long-term survival. Endothelial cell-specific deletion of Flrt2 in mice selectively pruned abnormalized vessels, resulting in a unique metabolic state termed "oxygen-glucose uncoupling," which suppressed tumor metastasis. Moreover, Flrt2 deletion caused an increase in the number of mature vessels, resulting in a significant increase in the antitumor effects of immune checkpoint blockers. Mechanistically, we found that FLRT2 forms noncanonical interendothelial adhesions that safeguard against oxidative stress through homophilic binding. Together, our results demonstrated the existence of tumor-specific interendothelial adhesions that enable abnormalized vessels to facilitate cancer aggressiveness. Targeting this type of adhesion complex could be a safe and effective therapeutic option to suppress cancer progression.

Preserving Vascular Integrity Protects Mice against Multidrug-Resistant Gram-Negative Bacterial Infection.

The rise in multidrug-resistant (MDR) organisms portends a serious global threat to the health care system with nearly untreatable infectious diseases, including pneumonia and its often fatal sequelae, acute respiratory distress syndrome (ARDS) and sepsis. Gram-negative bacteria (GNB), including Acinetobacter baumannii, Pseudomonas aeruginosa, and carbapenemase-producing Klebsiella pneumoniae (CPKP), are among the World Health Organization's and National Institutes of Health's high-priority MDR pathogens for targeted development of new therapies. Here, we show that stabilizing the host's vasculature by genetic deletion or pharmacological inhibition of the small GTPase ADP-ribosylation factor 6 (ARF6) increases survival rates of mice infected with A. baumannii, P. aeruginosa, and CPKP. We show that the pharmacological inhibition of ARF6-GTP phenocopies endothelium-specific Arf6 disruption in enhancing the survival of mice with A. baumannii pneumonia, suggesting that inhibition is on target. Finally, we show that the mechanism of protection elicited by these small-molecule inhibitors acts by the restoration of vascular integrity disrupted by GNB lipopolysaccharide (LPS) activation of the TLR4/MyD88/ARNO/ARF6 pathway. By targeting the host's vasculature with small-molecule inhibitors of ARF6 activation, we circumvent microbial drug resistance and provide a potential alternative/adjunctive treatment for emerging and reemerging pathogens.

Tris DBA palladium is an orally available inhibitor of GNAQ mutant uveal melanoma in vivo.

Uveal melanoma is a rare but often lethal malignancy and is the leading cause of death due to an ophthalmic condition. Uveal melanoma is often diagnosed at a late stage and has a strong propensity to hepatic metastasis. Recently, the most common driver mutations in uveal melanoma have been identified, predominantly in the G-proteins GNAQ. This pattern differs from that of cutaneous melanoma in which Braf and Nras predominate. There are no current clinically used agents that target GNAQ mutations, unlike the use of Braf inhibitors in cutaneous melanoma. We tested the novel agent Tris DBA palladium and found that it was markedly more effective against GNAQ mutant melanomas than wild type uveal melanomas. Given that ARF6 has recently been discovered as a node in GNAQ mutations, we evaluated the efficacy of Tris DBA palladium on ARF6 signaling and found that it was effective in inhibiting ARF6 activation. Finally, Tris DBA palladium was orally effective against GNAQ mutant melanoma in vivo. Tris DBA Palladium deserves further evaluation as a systemic agent for uveal melanoma.

The Small GTPase ARF6 Activates PI3K in Melanoma to Induce a Prometastatic State.

Melanoma has an unusual capacity to spread in early-stage disease, prompting aggressive clinical intervention in very thin primary tumors. Despite these proactive efforts, patients with low-risk, low-stage disease can still develop metastasis, indicating the presence of permissive cues for distant spread. Here, we show that constitutive activation of the small GTPase ARF6 (ARF6Q67L) is sufficient to accelerate metastasis in mice with BRAFV600E/Cdkn2aNULL melanoma at a similar incidence and severity to Pten loss, a major driver of PI3K activation and melanoma metastasis. ARF6Q67L promoted spontaneous metastasis from significantly smaller primary tumors than PTENNULL, implying an enhanced ability of ARF6-GTP to drive distant spread. ARF6 activation increased lung colonization from circulating melanoma cells, suggesting that the prometastatic function of ARF6 extends to late steps in metastasis. Unexpectedly, ARF6Q67L tumors showed upregulation of Pik3r1 expression, which encodes the p85 regulatory subunit of PI3K. Tumor cells expressing ARF6Q67L displayed increased PI3K protein levels and activity, enhanced PI3K distribution to cellular protrusions, and increased AKT activation in invadopodia. ARF6 is necessary and sufficient for activation of both PI3K and AKT, and PI3K and AKT are necessary for ARF6-mediated invasion. We provide evidence for aberrant ARF6 activation in human melanoma samples, which is associated with reduced survival. Our work reveals a previously unknown ARF6-PI3K-AKT proinvasive pathway, it demonstrates a critical role for ARF6 in multiple steps of the metastatic cascade, and it illuminates how melanoma cells can acquire an early metastatic phenotype in patients. SIGNIFICANCE: These findings reveal a prometastatic role for ARF6 independent of tumor growth, which may help explain how melanoma spreads distantly from thin, early-stage primary tumors.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/11/2892/F1.large.jpg.

Deletion of Robo4 prevents high-fat diet-induced adipose artery and systemic metabolic dysfunction.

OBJECTIVE: Accumulating evidence suggests the vascular endothelium plays a fundamental role in the pathophysiology of obesity by regulating the functional status of white adipose and systemic metabolism. Robo4 is expressed specifically in endothelial cells and increases vascular stability and inhibits angiogenesis. We sought to determine the role of Robo4 in modulating cardiometabolic function in response to high-fat feeding. METHODS: We examined exercise capacity, glucose tolerance, and white adipose tissue artery gene expression, endothelium-dependent dilation (EDD), and angiogenesis in wild type and Robo4 knockout (KO) mice fed normal chow (NC) or a high-fat diet (HFD). RESULTS: We found Robo4 deletion enhances exercise capacity in NC-fed mice and HFD markedly increased the expression of the Robo4 ligand, Slit2, in white adipose tissue. Deletion of Robo4 increased angiogenesis in white adipose tissue and protected against HFD-induced impairments in white adipose artery vasodilation and glucose intolerance. CONCLUSIONS: We demonstrate a novel functional role for Robo4 in endothelial cell function and metabolic homeostasis in white adipose tissue, with Robo4 deletion protecting against endothelial and metabolic dysfunction associated with a HFD. Our findings suggest that Robo4-dependent signaling pathways may be a novel target in anti-obesity therapy.

Cerebral and skeletal muscle feed artery vasoconstrictor responses in a mouse model with greater large elastic artery stiffness.

NEW FINDINGS: What is the central question of this study? Greater large artery stiffness is associated with dysfunctional resistance artery vasodilatory responses, impaired memory and greater risk of Alzheimer's disease. However, it is unknown whether stiffer large arteries affect cerebral and skeletal muscle feed artery responses to vasoconstrictors. What is the main finding and its importance? In a mouse model with greater large artery stiffness (Eln+/- ), we find an exacerbated vasoconstrictor response to angiotensin II in cerebral arteries, but not skeletal muscle feed arteries, thus implicating altered cerebral artery angiotensin II responsiveness in the poor brain outcomes associated with greater large artery stiffness. ABSTRACT: Greater stiffness of the large elastic arteries is associated with end-organ damage and dysfunction. At the same time, resistance artery vasoconstrictor responsiveness influences vascular tone and organ blood flow. However, it is unknown whether large elastic artery stiffness modulates the responsiveness to vasoconstrictors in resistance arteries of the cerebral or skeletal muscle circulations. We previously described the elastin haploinsufficient (Eln+/- ) mouse as a model with greater aortic stiffness, but with similar cerebral and skeletal muscle feed artery stiffness to wild-type (Eln+/+ ) mice. Here, we used this model to examine the relationship between large elastic artery stiffness and resistance artery vasoconstrictor responses. In middle cerebral arteries (MCAs), vasoconstriction in response to angiotensin II (Ang II) was ∼40% greater in Eln+/- compared with Eln+/+ mice (P = 0.02), and this group difference was ameliorated by losartan, indicating a role for Ang II type 1 receptors (AT1Rs). In gastrocnemius feed arteries, Eln+/- and Eln+/+ mice did not differ in the response to Ang II. In addition, the vasoconstrictor responses to noradrenaline, endothelin-1 and potassium chloride were not different between Eln+/- and Eln+/+ mice for either MCAs or gastrocnemius feed arteries. The MCA AT1R gene expression did not differ between groups, whereas Ang II type 2 receptor gene expression was ∼50% lower in MCAs from Eln+/- versus Eln+/+ mice (P = 0.01). In conclusion, greater large elastic artery stiffness is associated with an exacerbated vasoconstriction response to Ang II in cerebral arteries, but is not associated with the responses to other vasoconstrictors in either cerebral or skeletal muscle feed arteries.

The small GTPase ARF6 regulates protein trafficking to control cellular function during development and in disease.

The activation of the small GTPase ARF6 has been implicated in promoting several pathological processes related to vascular instability and tumor formation, growth, and metastasis. ARF6 also plays a vital role during embryonic development. Recent studies have suggested that ARF6 carries out these disparate functions primarily by controlling protein trafficking within the cell. ARF6 helps direct proteins to intracellular or extracellular locations where they function in normal cellular responses during development and in pathological processes later in life. This transport of proteins is accomplished through a variety of mechanisms, including endocytosis and recycling, microvesicle release, and as yet uncharacterized processes. This Commentary will explore the functions of ARF6, while focusing on the role of this small GTPase in development and postnatal physiology, regulating barrier function and diseases associated with its loss, and tumor formation, growth, and metastasis.

PI3Ka-Akt1-mediated Prdm4 induction in adipose tissue increases energy expenditure, inhibits weight gain, and improves insulin resistance in diet-induced obese mice.

Stimulation of white adipose tissue (WAT) browning is considered as a potential approach to treat obesity and metabolic diseases. Our previous studies have shown that phytochemical butein can stimulate WAT browning through induction of Prdm4 in adipocytes. Here, we investigated the effects of butein on diet-induced obesity and its underlying molecular mechanism. Treatment with butein prevented weight gains and improved metabolic profiles in diet-induced obese mice. Butein treatment groups also displayed higher body temperature, increased energy expenditure, and enhanced expression of thermogenic genes in adipose tissue. Butein also suppressed body weight gains and improved glucose and insulin tolerance in mice housed at thermoneutrality (30 °C). These effects were associated with adipose-selective induction of Prdm4, suggesting the role of Prdm4 in butein-mediated anti-obese effects. To directly assess the in vivo role of Prdm4, we generated aP2-Prdm4 transgenic mouse lines overexpressing Prdm4 in adipose tissues. Adipose-specific transgenic expression of Prdm4 recapitulated the butein's actions in stimulating energy expenditure, cold tolerance, and thermogenic gene expression, resulting in prevention of obesity and improvement of metabolism. Mechanistically, direct inhibition of PI3Kα activity followed by selective suppression of its downstream Akt1 mirrored butein's effect on Ucp1 expression and oxygen consumption. In addition, effects of butein were completely abolished in Akt1 KO mouse embryonic fibroblasts. Together, these studies demonstrate the role of butein in obesity and metabolic diseases, further highlighting that adipose PI3Kα-Akt1-Prdm4 axis is a regulator of energy expenditure.

Plasma Biomarkers of Inflammation and Angiogenesis Predict Cerebral Cavernous Malformation Symptomatic Hemorrhage or Lesional Growth.

RATIONALE: The clinical course of cerebral cavernous malformations is highly unpredictable, with few cross-sectional studies correlating proinflammatory genotypes and plasma biomarkers with prior disease severity. OBJECTIVE: We hypothesize that a panel of 24 candidate plasma biomarkers, with a reported role in the physiopathology of cerebral cavernous malformations, may predict subsequent clinically relevant disease activity. METHODS AND RESULTS: Plasma biomarkers were assessed in nonfasting peripheral venous blood collected from consecutive cerebral cavernous malformation subjects followed for 1 year after initial sample collection. A first cohort (N=49) was used to define the best model of biomarker level combinations to predict a subsequent symptomatic lesional hemorrhagic expansion within a year after the blood sample. We generated the receiver operating characteristic curves and area under the curve for each biomarker individually and each weighted linear combination of relevant biomarkers. The best model to predict lesional activity was selected as that minimizing the Akaike information criterion. In this cohort, 11 subjects experienced symptomatic lesional hemorrhagic expansion (5 bleeds and 10 lesional growths) within a year after the blood draw. Subjects had lower soluble CD14 (cluster of differentiation 14; P=0.05), IL (interleukin)-6 (P=0.04), and VEGF (vascular endothelial growth factor; P=0.0003) levels along with higher plasma levels of IL-1ß (P=0.008) and soluble ROBO4 (roundabout guidance receptor 4; P=0.03). Among the 31 weighted linear combinations of these 5 biomarkers, the best model (with the lowest Akaike information criterion value, 25.3) was the weighted linear combination including soluble CD14, IL-1ß, VEGF, and soluble ROBO4, predicting a symptomatic hemorrhagic expansion with a sensitivity of 86% and specificity of 88% (area under the curve, 0.90; P<0.0001). We then validated our best model in the second sequential independent cohort (N=28). CONCLUSIONS: This is the first study reporting a predictive association between plasma biomarkers and subsequent cerebral cavernous malformation disease clinical activity. This may be applied in clinical prognostication and stratification of cases in clinical trials.

Publisher Correction: Combating subclonal evolution of resistant cancer phenotypes.

The originally published version of this Article contained an error in Figure 4. In panel a, grey boxes surrounding the subclones associated with patients #2 and #4 obscured adjacent portions of the heatmap. This error has now been corrected in both the PDF and HTML versions of the Article.

Right ventricular involution: What can we learn from nature's model of compensated hypertrophy?

BACKGROUND: Right ventricular (RV) failure (RVF) is a vexing problem facing patients with various disease processes and carries a high mortality. RVF is a poorly understood phenomenon with limited treatment options. In mammalian fetal circulation, the right ventricle is the systemic ventricle. In neonates, however, the left ventricle assumes that role and gradually thickens compared with the right ventricle. This process, known as right ventricular involution (RVI), is poorly understood. We sought to define the time course and identify mechanisms involved in RVI. METHODS: Wild-type mice were bred and sacrificed on day of life (DOL) 1, 4, 8, 16, and 30 to evaluate left ventricular (LV) and RV wall thickness and apoptosis. A terminal deoxynucleotidyl transferase nick-end labeling assay and RNA sequencing were performed to measure changes during RVI. RESULTS: Morphometric analysis demonstrated the changes in RV and LV wall thickness occurring between DOL 1 and DOL 16 (RV:LV, 0.53:0.44; P = .03). In addition, apoptosis was most active early, with the highest percentage of apoptotic cells on DOL 1 (1.0%) and a significant decrease by DOL 30 (0.23%) (P = .02). Similarly, expression of the proapoptotic genes BCL2l11 and Pawr were increased at DOL 1, and the antiapoptotic genes Nol3 and Naip2 were significantly increased at DOL 30. CONCLUSIONS: RVI is a misnomer, but significant changes occur early (by DOL 16) in neonatal mouse hearts. Apoptosis plays a role in RVI, but whether manipulation of apoptotic pathways can prevent or reverse RVI is unknown and warrants further investigation.

Plasma Biomarkers of Inflammation Reflect Seizures and Hemorrhagic Activity of Cerebral Cavernous Malformations.

The clinical course of cerebral cavernous malformations (CCMs) is highly variable. Based on recent discoveries implicating angiogenic and inflammatory mechanisms, we hypothesized that serum biomarkers might reflect chronic or acute disease activity. This single-site prospective observational cohort study included 85 CCM patients, in whom 24 a priori chosen plasma biomarkers were quantified and analyzed in relation to established clinical and imaging parameters of disease categorization and severity. We subsequently validated the positive correlations in longitudinal follow-up of 49 subjects. Plasma levels of matrix metalloproteinase-2 and intercellular adhesion molecule 1 were significantly higher (P = 0.02 and P = 0.04, respectively, FDR corrected), and matrix metalloproteinase-9 was lower (P = 0.04, FDR corrected) in patients with seizure activity at any time in the past. Vascular endothelial growth factor and endoglin (both P = 0.04, FDR corrected) plasma levels were lower in patients who had suffered a symptomatic bleed in the prior 3 months. The hierarchical clustering analysis revealed a cluster of four plasma inflammatory cytokines (interleukin 2, interferon gamma, tumor necrosis factor alpha, and interleukin 1 beta) separating patients into what we designated "high" and "low" inflammatory states. The "high" inflammatory state was associated with seizure activity (P = 0.02) and more than one hemorrhagic event during a patient's lifetime (P = 0.04) and with a higher rate of new hemorrhage, lesion growth, or new lesion formation (P < 0.05) during prospective follow-up. Peripheral plasma biomarkers reflect seizure and recent hemorrhagic activity in CCM patients. In addition, four clustered inflammatory biomarkers correlate with cumulative disease aggressiveness and predict future clinical activity.

Combating subclonal evolution of resistant cancer phenotypes.

Metastatic breast cancer remains challenging to treat, and most patients ultimately progress on therapy. This acquired drug resistance is largely due to drug-refractory sub-populations (subclones) within heterogeneous tumors. Here, we track the genetic and phenotypic subclonal evolution of four breast cancers through years of treatment to better understand how breast cancers become drug-resistant. Recurrently appearing post-chemotherapy mutations are rare. However, bulk and single-cell RNA sequencing reveal acquisition of malignant phenotypes after treatment, including enhanced mesenchymal and growth factor signaling, which may promote drug resistance, and decreased antigen presentation and TNF-α signaling, which may enable immune system avoidance. Some of these phenotypes pre-exist in pre-treatment subclones that become dominant after chemotherapy, indicating selection for resistance phenotypes. Post-chemotherapy cancer cells are effectively treated with drugs targeting acquired phenotypes. These findings highlight cancer's ability to evolve phenotypically and suggest a phenotype-targeted treatment strategy that adapts to cancer as it evolves.

Small GTPase ARF6 controls VEGFR2 trafficking and signaling in diabetic retinopathy.

The devastating sequelae of diabetes mellitus include microvascular permeability, which results in retinopathy. Despite clinical and scientific advances, there remains a need for new approaches to treat retinopathy. Here, we have presented a possible treatment strategy, whereby targeting the small GTPase ARF6 alters VEGFR2 trafficking and reverses signs of pathology in 4 animal models that represent features of diabetic retinopathy and in a fifth model of ocular pathological angiogenesis. Specifically, we determined that the same signaling pathway utilizes distinct GEFs to sequentially activate ARF6, and these GEFs exert distinct but complementary effects on VEGFR2 trafficking and signal transduction. ARF6 activation was independently regulated by 2 different ARF GEFs - ARNO and GEP100. Interaction between VEGFR2 and ARNO activated ARF6 and stimulated VEGFR2 internalization, whereas a VEGFR2 interaction with GEP100 activated ARF6 to promote VEGFR2 recycling via coreceptor binding. Intervening in either pathway inhibited VEGFR2 signal output. Finally, using a combination of in vitro, cellular, genetic, and pharmacologic techniques, we demonstrated that ARF6 is pivotal in VEGFR2 trafficking and that targeting ARF6-mediated VEGFR2 trafficking has potential as a therapeutic approach for retinal vascular diseases such as diabetic retinopathy.

Calcium influx through TRPV4 channels modulates the adherens contacts between retinal microvascular endothelial cells.

KEY POINTS: Endothelial cells employ transient receptor potential isoform 4 (TRPV4) channels to sense ambient mechanical and chemical stimuli. In retinal microvascular endothelial cells, TRPV4 channels regulate calcium homeostasis, cytoskeletal signalling and the organization of adherens junctional contacts. Intracellular calcium increases induced by TRPV4 agonists include a significant contribution from calcium release from internal stores. Activation of TRPV4 channels regulates retinal endothelial barriers in vitro and in vivo. TRPV4 sensing may provide a feedback mechanism between sensing shear flow and eicosanoid modulators, vascular permeability and contractility at the inner retinal endothelial barrier. ABSTRACT: The identity of microvascular endothelial (MVE) mechanosensors that sense blood flow in response to mechanical and chemical stimuli and regulate vascular permeability in the retina is unknown. Using immunohistochemistry, calcium imaging, electrophysiology, impedance measurements and vascular permeability assays, we show that the transient receptor potential isoform 4 (TRPV4) plays a major role in Ca2+ /cation signalling, cytoskeletal remodelling and barrier function in retinal microvasculature in vitro and in vivo. Human retinal MVE cells (HrMVECs) predominantly expressed Trpv1 and Trpv4 transcripts, and TRPV4 was broadly localized to the plasma membrane of cultured cells and intact blood vessels in the inner retina. Treatment with the selective TRPV4 agonist GSK1016790A (GSK101) activated a nonselective cation current, robustly elevated [Ca2+ ]i and reversibly increased the permeability of MVEC monolayers. This was associated with disrupted organization of endothelial F-actin, downregulated expression of occludin and remodelling of adherens contacts consisting of vascular endothelial cadherin (VE-cadherin) and ß-catenin. In vivo, GSK101 increased the permeability of retinal blood vessels in wild type but not in TRPV4 knockout mice. Agonist-evoked effects on barrier permeability and cytoskeletal reorganization were antagonized by the selective TRPV4 blocker HC 067047. Human choroidal endothelial cells expressed lower TRPV4 mRNA/protein levels and showed less pronounced agonist-evoked calcium signals compared to MVECs. These findings indicate a major role for TRPV4 in Ca2+ homeostasis and barrier function in human retinal capillaries and suggest that TRPV4 may differentially contribute to the inner vs. outer blood-retinal barrier function.

Control of intestinal stem cell function and proliferation by mitochondrial pyruvate metabolism.

Most differentiated cells convert glucose to pyruvate in the cytosol through glycolysis, followed by pyruvate oxidation in the mitochondria. These processes are linked by the mitochondrial pyruvate carrier (MPC), which is required for efficient mitochondrial pyruvate uptake. In contrast, proliferative cells, including many cancer and stem cells, perform glycolysis robustly but limit fractional mitochondrial pyruvate oxidation. We sought to understand the role this transition from glycolysis to pyruvate oxidation plays in stem cell maintenance and differentiation. Loss of the MPC in Lgr5-EGFP-positive stem cells, or treatment of intestinal organoids with an MPC inhibitor, increases proliferation and expands the stem cell compartment. Similarly, genetic deletion of the MPC in Drosophila intestinal stem cells also increases proliferation, whereas MPC overexpression suppresses stem cell proliferation. These data demonstrate that limiting mitochondrial pyruvate metabolism is necessary and sufficient to maintain the proliferation of intestinal stem cells.

Induction of thermogenic adipocytes: molecular targets and thermogenic small molecules.

Adipose tissue is a central metabolic organ that controls energy homeostasis of the whole body. White adipose tissue (WAT) stores excess energy in the form of triglycerides, whereas brown adipose tissue (BAT) dissipates energy in the form of heat through mitochondrial uncoupling protein 1 (Ucp1). A newly identified adipose tissue called 'beige fat' (BAT-like) is produced through a process called WAT browning. This tissue mainly resides in WAT depots and displays intermediate characteristics of both WAT and BAT. Since the recent discovery of BAT in the human body, along with the identification of molecular targets for BAT activation, stimulating energy expenditure has been considered as a great strategy to treat human obesity and metabolic diseases. Here we summarize recent findings regarding molecular targets and thermogenic small molecules that can stimulate BAT and increase energy expenditure, with an emphasis on possible therapeutic applications in humans.

Placental labyrinth formation in mice requires endothelial FLRT2/UNC5B signaling.

The placental labyrinth is the interface for gas and nutrient exchange between the embryo and the mother; hence its proper development is essential for embryogenesis. However, the molecular mechanism underlying development of the placental labyrinth, particularly in terms of its endothelial organization, is not well understood. Here, we determined that fibronectin leucine-rich transmembrane protein 2 (FLRT2), a repulsive ligand of the UNC5 receptor family for neurons, is unexpectedly expressed in endothelial cells specifically in the placental labyrinth. Mice lacking FLRT2 in endothelial cells exhibited embryonic lethality at mid-gestation, with systemic congestion and hypoxia. Although they lacked apparent deformities in the embryonic vasculature and heart, the placental labyrinths of these embryos exhibited aberrant alignment of endothelial cells, which disturbed the feto-maternal circulation. Interestingly, this vascular deformity was related to endothelial repulsion through binding to the UNC5B receptor. Our results suggest that the proper organization of the placental labyrinth depends on coordinated inter-endothelial repulsion, which prevents uncontrolled layering of the endothelium.

Endothelial TLR4 and the microbiome drive cerebral cavernous malformations.

Cerebral cavernous malformations (CCMs) are a cause of stroke and seizure for which no effective medical therapies yet exist. CCMs arise from the loss of an adaptor complex that negatively regulates MEKK3-KLF2/4 signalling in brain endothelial cells, but upstream activators of this disease pathway have yet to be identified. Here we identify endothelial Toll-like receptor 4 (TLR4) and the gut microbiome as critical stimulants of CCM formation. Activation of TLR4 by Gram-negative bacteria or lipopolysaccharide accelerates CCM formation, and genetic or pharmacologic blockade of TLR4 signalling prevents CCM formation in mice. Polymorphisms that increase expression of the TLR4 gene or the gene encoding its co-receptor CD14 are associated with higher CCM lesion burden in humans. Germ-free mice are protected from CCM formation, and a single course of antibiotics permanently alters CCM susceptibility in mice. These studies identify unexpected roles for the microbiome and innate immune signalling in the pathogenesis of a cerebrovascular disease, as well as strategies for its treatment.