GPR124 regulates murine brain embryonic angiogenesis and BBB formation by an intracellular domain-independent mechanism.

The GPR124/RECK/WNT7 pathway is an essential regulator of CNS angiogenesis and blood-brain barrier (BBB) function. GPR124, a brain endothelial adhesion seven-pass transmembrane protein, associates with RECK, which binds and stabilizes newly synthesized WNT7 that is transferred to frizzled (FZD) to initiate canonical ß-catenin signaling. GPR124 remains enigmatic: although its extracellular domain (ECD) is essential, the poorly conserved intracellular domain (ICD) appears to be variably required in mammals versus zebrafish, potentially via adaptor protein bridging of GPR124 and FZD ICDs. GPR124 ICD deletion impairs zebrafish angiogenesis, but paradoxically retains WNT7 signaling upon mammalian transfection. We thus investigated GPR124 ICD function using the mouse deletion mutant Gpr124ΔC. Despite inefficiently expressed GPR124ΔC protein, Gpr124ΔC/ΔC mice could be born with normal cerebral cortex angiogenesis, in comparison with Gpr124-/- embryonic lethality, forebrain avascularity and hemorrhage. Gpr124ΔC/ΔC vascular phenotypes were restricted to sporadic ganglionic eminence angiogenic defects, attributable to impaired GPR124ΔC protein expression. Furthermore, Gpr124ΔC and the recombinant GPR124 ECD rescued WNT7 signaling in culture upon brain endothelial Gpr124 knockdown. Thus, in mice, GPR124-regulated CNS forebrain angiogenesis and BBB function are exerted by ICD-independent functionality, extending the signaling mechanisms used by adhesion seven-pass transmembrane receptors.

PIK3CA and CCM mutations fuel cavernomas through a cancer-like mechanism.

Vascular malformations are thought to be monogenic disorders that result in dysregulated growth of blood vessels. In the brain, cerebral cavernous malformations (CCMs) arise owing to inactivation of the endothelial CCM protein complex, which is required to dampen the activity of the kinase MEKK31-4. Environmental factors can explain differences in the natural history of CCMs between individuals5, but why single CCMs often exhibit sudden, rapid growth, culminating in strokes or seizures, is unknown. Here we show that growth of CCMs requires increased signalling through the phosphatidylinositol-3-kinase (PI3K)-mTOR pathway as well as loss of function of the CCM complex. We identify somatic gain-of-function mutations in PIK3CA and loss-of-function mutations in the CCM complex in the same cells in a majority of human CCMs. Using mouse models, we show that growth of CCMs requires both PI3K gain of function and CCM loss of function in endothelial cells, and that both CCM loss of function and increased expression of the transcription factor KLF4 (a downstream effector of MEKK3) augment mTOR signalling in endothelial cells. Consistent with these findings, the mTORC1 inhibitor rapamycin effectively blocks the formation of CCMs in mouse models. We establish a three-hit mechanism analogous to cancer, in which aggressive vascular malformations arise through the loss of vascular 'suppressor genes' that constrain vessel growth and gain of a vascular 'oncogene' that stimulates excess vessel growth. These findings suggest that aggressive CCMs could be treated using clinically approved mTORC1 inhibitors.

Cell-autonomous requirement for ACE2 across organs in lethal mouse SARS-CoV-2 infection.

Angiotensin-converting enzyme 2 (ACE2) is the cell-surface receptor for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). While its central role in Coronavirus Disease 2019 (COVID-19) pathogenesis is indisputable, there remains significant debate regarding the role of this transmembrane carboxypeptidase in the disease course. These include the role of soluble versus membrane-bound ACE2, as well as ACE2-independent mechanisms that may contribute to viral spread. Testing these roles requires in vivo models. Here, we report humanized ACE2-floxed mice in which hACE2 is expressed from the mouse Ace2 locus in a manner that confers lethal disease and permits cell-specific, Cre-mediated loss of function, and LSL-hACE2 mice in which hACE2 is expressed from the Rosa26 locus enabling cell-specific, Cre-mediated gain of function. Following exposure to SARS-CoV-2, hACE2-floxed mice experienced lethal cachexia, pulmonary infiltrates, intravascular thrombosis and hypoxemia-hallmarks of severe COVID-19. Cre-mediated loss and gain of hACE2 demonstrate that neuronal infection confers lethal cachexia, hypoxemia, and respiratory failure in the absence of lung epithelial infection. In this series of genetic experiments, we demonstrate that ACE2 is absolutely and cell-autonomously required for SARS-CoV-2 infection in the olfactory epithelium, brain, and lung across diverse cell types. Therapies inhibiting or blocking ACE2 at these different sites are likely to be an effective strategy towards preventing severe COVID-19.

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.

Cerebral cavernous malformations arise from endothelial gain of MEKK3-KLF2/4 signalling.

Cerebral cavernous malformations (CCMs) are common inherited and sporadic vascular malformations that cause strokes and seizures in younger individuals. CCMs arise from endothelial cell loss of KRIT1, CCM2 or PDCD10, non-homologous proteins that form an adaptor complex. How disruption of the CCM complex results in disease remains controversial, with numerous signalling pathways (including Rho, SMAD and Wnt/ß-catenin) and processes such as endothelial-mesenchymal transition (EndMT) proposed to have causal roles. CCM2 binds to MEKK3 (refs 7, 8, 9, 10, 11), and we have recently shown that CCM complex regulation of MEKK3 is essential during vertebrate heart development. Here we investigate this mechanism in CCM disease pathogenesis. Using a neonatal mouse model of CCM disease, we show that expression of the MEKK3 target genes Klf2 and Klf4, as well as Rho and ADAMTS protease activity, are increased in the endothelial cells of early CCM lesions. By contrast, we find no evidence of EndMT or increased SMAD or Wnt signalling during early CCM formation. Endothelial-specific loss of Map3k3 (also known as Mekk3), Klf2 or Klf4 markedly prevents lesion formation, reverses the increase in Rho activity, and rescues lethality. Consistent with these findings in mice, we show that endothelial expression of KLF2 and KLF4 is increased in human familial and sporadic CCM lesions, and that a disease-causing human CCM2 mutation abrogates the MEKK3 interaction without affecting CCM complex formation. These studies identify gain of MEKK3 signalling and KLF2/4 function as causal mechanisms for CCM pathogenesis that may be targeted to develop new CCM therapeutics.

Phenotypic characterization of murine models of cerebral cavernous malformations.

Cerebral cavernous malformations (CCMs) are clusters of dilated capillaries that affect around 0.5% of the population. CCMs exist in two forms, sporadic and familial. Mutations in three documented genes, KRIT1(CCM1), CCM2, and PDCD10(CCM3), cause the autosomal dominant form of the disease, and somatic mutations in these same genes underlie lesion development in the brain. Murine models with constitutive or induced loss of respective genes have been applied to study disease pathobiology and therapeutic manipulations. We aimed to analyze the phenotypic characteristic of two main groups of models, the chronic heterozygous models with sensitizers promoting genetic instability, and the acute neonatal induced homozygous knockout model. Acute model mice harbored a higher lesion burden than chronic models, more localized in the hindbrain, and largely lacking iron deposition and inflammatory cell infiltrate. The chronic model mice showed a lower lesion burden localized throughout the brain, with significantly greater perilesional iron deposition, immune B- and T-cell infiltration, and less frequent junctional protein immunopositive endothelial cells. Lesional endothelial cells in both models expressed similar phosphorylated myosin light chain immunopositivity indicating Rho-associated protein kinase activity. These data suggest that acute models are better suited to study the initial formation of the lesion, while the chronic models better reflect lesion maturation, hemorrhage, and inflammatory response, relevant pathobiologic features of the human disease.

Cerebral cavernous malformations arise independent of the heart of glass receptor.

BACKGROUND AND PURPOSE: The Heart of Glass (HEG) receptor binds KRIT1 and functions with KRIT1, CCM2, and PDCD10 in a common signaling pathway required for heart and vascular development. Mutations in KRIT1, CCM2, and PDCD10 also underlie human cerebral cavernous malformation (CCM) and postnatal loss of these genes in the mouse endothelium results in rapid CCM formation. Here, we test the role of HEG in CCM formation in mice and in humans. METHODS: We constitutively or conditionally deleted Heg and Ccm2 genes in genetically modified mice. Mouse embryos, brain, and retina tissues were analyzed to assess CCM lesion formation. RESULTS: In postnatal mice, CCMs form with Ccm2-/- but not with Heg-/- or Heg-/-;Ccm2+/- endothelial cells. Consistent with these findings, human patients with CCM who lack exonic mutations in KRIT1, CCM2, or PDCD10 do not have mutations in HEG. CONCLUSIONS: These findings suggest that the HEG-CCM signaling functions during cardiovascular development and growth, whereas CCMs arise because of loss of HEG-independent CCM signaling in the endothelium of the central nervous system after birth.

Endothelial SARS-CoV-2 infection is not the underlying cause of COVID-19-associated vascular pathology in mice.

Endothelial damage and vascular pathology have been recognized as major features of COVID-19 since the beginning of the pandemic. Two main theories regarding how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) damages endothelial cells and causes vascular pathology have been proposed: direct viral infection of endothelial cells or indirect damage mediated by circulating inflammatory molecules and immune mechanisms. However, these proposed mechanisms remain largely untested in vivo. In the present study, we utilized a set of new mouse genetic tools developed in our lab to test both the necessity and sufficiency of endothelial human angiotensin-converting enzyme 2 (hACE2) in COVID-19 pathogenesis. Our results demonstrate that endothelial ACE2 and direct infection of vascular endothelial cells do not contribute significantly to the diverse vascular pathology associated with COVID-19.

Endothelial SARS-CoV-2 infection is not the underlying cause of COVID19-associated vascular pathology in mice.

Endothelial damage and vascular pathology have been recognized as major features of COVID-19 since the beginning of the pandemic. Two main theories regarding how Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) damages endothelial cells and causes vascular pathology have been proposed: direct viral infection of endothelial cells or indirect damage mediated by circulating inflammatory molecules and immune mechanisms. However, these proposed mechanisms remain largely untested in vivo. Here, we utilized a set of new mouse genetic tools 1 developed in our lab to test both the necessity and sufficiency of endothelial human angiotensin-converting enzyme 2 (hACE2) in COVID19 pathogenesis. Our results demonstrate that endothelial ACE2 and direct infection of vascular endothelial cells does not contribute significantly to the diverse vascular pathology associated with COVID-19.

Mouse placenta fetal macrophages arise from endothelial cells outside the placenta.

Placental fetal macrophages (fMacs) are the only immune cells on the fetal side of the placental barrier. Mouse models have not been used to test their function because they have previously been found to have distinct cellular origins and functions in mice and humans. Here, we test the ontogeny of mouse placental fMacs. Using a new Hoxa13Cre allele that labels all placental endothelial cells (ECs), we demonstrate that mouse placenta fMacs do not arise from placental endothelium. Instead, lineage tracing studies using Tie2-Cre and Cx3cr1CreERT2 alleles demonstrate that mouse placental fMacs arise from yolk sac endothelium. Administration of blocking antibodies against CSF1R at E6.5 and E7.5 results in depletion of placental fMacs throughout pregnancy, and this suggests a yolk sac origin, similar to that in human fMacs. This Matters Arising paper is in response to Liang et al., published in Developmental Cell. A response by Liang and Liu is published in this issue.

Sinusoidal and lymphatic vessel growth is controlled by reciprocal VEGF-C-CDH5 inhibition.

Sinusoids are specialized, low pressure blood vessels in the liver, bone marrow, and spleen required for definitive hematopoiesis. Unlike other blood endothelial cells (ECs), sinusoidal ECs express high levels of VEGFR3. VEGFR3 and its ligand VEGF-C are known to support lymphatic growth, but their function in sinusoidal vessels is unknown. In this study, we define a reciprocal VEGF-C/VEGFR3-CDH5 (VE-cadherin) signaling axis that controls growth of both sinusoidal and lymphatic vessels. Loss of VEGF-C or VEGFR3 resulted in cutaneous edema, reduced fetal liver size, and bloodless bone marrow due to impaired lymphatic and sinusoidal vessel growth. Mice with membrane-retained VE-cadherin conferred identical lymphatic and sinusoidal defects, suggesting that VE-cadherin opposes VEGF-C/VEGFR3 signaling. In developing mice, loss of VE-cadherin rescued defects in sinusoidal and lymphatic growth caused by loss of VEGFR3 but not loss of VEGF-C, findings explained by potentiated VEGF-C/VEGFR2 signaling in VEGFR3-deficient lymphatic ECs. Mechanistically, VEGF-C/VEGFR3 signaling induces VE-cadherin endocytosis and loss of function via SRC-mediated phosphorylation, while VE-cadherin prevents VEGFR3 endocytosis required for optimal receptor signaling. These findings establish an essential role for VEGF-C/VEGFR3 signaling during sinusoidal vascular growth, identify VE-cadherin as a powerful negative regulator of VEGF-C signaling that acts through both VEGFR3 and VEGFR2 receptors, and suggest that negative regulation of VE-cadherin is required for effective VEGF-C/VEGFR3 signaling during growth of sinusoidal and lymphatic vessels. Manipulation of this reciprocal negative regulatory mechanism, e.g. by reducing VE-cadherin function, may be used to stimulate therapeutic sinusoidal or lymphatic vessel growth.

Genetic blockade of lymphangiogenesis does not impair cardiac function after myocardial infarction.

In recent decades, treatments for myocardial infarction (MI), such as stem and progenitor cell therapy, have attracted considerable scientific and clinical attention but failed to improve patient outcomes. These efforts indicate that more rigorous mechanistic and functional testing of potential MI therapies is required. Recent studies have suggested that augmenting post-MI lymphatic growth via VEGF-C administration improves cardiac function. However, the mechanisms underlying this proposed therapeutic approach remain vague and untested. To more rigorously test the role of lymphatic vessel growth after MI, we examined the post-MI cardiac function of mice in which lymphangiogenesis had been blocked genetically by pan-endothelial or lymphatic endothelial loss of the lymphangiogenic receptor VEGFR3 or global loss of the VEGF-C and VEGF-D ligands. The results obtained using all 3 genetic approaches were highly concordant and demonstrated that loss of lymphatic vessel growth did not impair left ventricular ejection fraction 2 weeks after MI in mice. We observed a trend toward excess fluid in the infarcted region of the left ventricle, but immune cell infiltration and clearance were unchanged with loss of expanded lymphatics. These studies refute the hypothesis that lymphangiogenesis contributes significantly to cardiac function after MI, and suggest that any effect of exogenous VEGF-C is likely to be mediated by nonlymphangiogenic mechanisms.

SARS-CoV-2 infection of olfactory epithelial cells and neurons drives acute lung injury and lethal COVID-19 in mice.

Lethal COVID-19 is associated with respiratory failure that is thought to be caused by acute respiratory distress syndrome (ARDS) secondary to pulmonary infection. To date, the cellular pathogenesis has been inferred from studies describing the expression of ACE2, a transmembrane protein required for SARS-CoV-2 infection, and detection of viral RNA or protein in infected humans, model animals, and cultured cells. To functionally test the cellular mechanisms of COVID-19, we generated hACE2 fl animals in which human ACE2 (hACE2) is expressed from the mouse Ace2 locus in a manner that permits cell-specific, Cre-mediated loss of function. hACE2 fl animals developed lethal weight loss and hypoxemia within 7 days of exposure to SARS-CoV-2 that was associated with pulmonary infiltrates, intravascular thrombosis and patchy viral infection of lung epithelial cells. Deletion of hACE2 in lung epithelial cells prevented viral infection of the lung, but not weight loss, hypoxemia or death. Inhalation of SARS-CoV-2 by hACE2 fl animals resulted in early infection of sustentacular cells with subsequent infection of neurons in the neighboring olfactory bulb and cerebral cortexâ€" events that did not require lung epithelial cell infection. Pharmacologic ablation of the olfactory epithelium or Foxg1 Cre mediated deletion of hACE2 in olfactory epithelial cells and neurons prevented lethality and neuronal infection following SARS-CoV-2 infection. Conversely, transgenic expression of hACE2 specifically in olfactory epithelial cells and neurons in Foxg1 Cre ; LSL- hACE2 mice was sufficient to confer neuronal infection associated with respiratory failure and death. These studies establish mouse loss and gain of function genetic models with which to genetically dissect viral-host interactions and demonstrate that lethal disease due to respiratory failure may arise from extrapulmonary infection of the olfactory epithelium and brain. Future therapeutic efforts focused on preventing olfactory epithelial infection may be an effective means of protecting against severe COVID-19.

Inhibition of carcinoma cell motility by epoxyeicosatrienoic acid (EET) antagonists.

Cytochrome P450 (CYP) epoxygenases, CYP2C8, 2C9 and 2J2 mRNA and proteins, were expressed in prostate carcinoma (PC-3, DU-145 and LNCaP) cells. 11,12-Epoxyeicosatrienoic acid (11,12-EET) was the major arachidonic acid metabolite in these cells. Blocking EET synthesis by a selective CYP epoxygenase inhibitor (N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide [MS-PPOH]) inhibited tonic (basal) invasion and migration (motility) while exogenously added EET induced cell motility in a concentration-dependent manner. An epidermal growth factor receptor (EGFR) kinase inhibitor (AG494) or a PI3 kinase inhibitor (LY294002) inhibited cell migration and reduced 11,12-EET-induced cell migration. Importantly, synthetic EET antagonists (14,15-epoxyeicosa-5(Z)-enoic acid [14,15-EEZE], 14,15-epoxyeicosa-5(Z)-enoic acid 2-[2-(3-hydroxy-propoxy)-ethoxy]-ethyl ester [14,15-EEZE-PEG] and 14,15-epoxyeicosa-5(Z)-enoic-methylsulfonylimide [14,15-EEZE-mSI]) inhibited EET-induced cell invasion and migration. 11,12-EET induced cell stretching and myosin-actin microfilament formation as well as increased phosphorylation of EGFR and Akt (Ser473), while 14,15-EEZE inhibited these effects. These results suggest that EET induce and EET antagonists inhibit cell motility, possibly by putative EET receptor-mediated EGFR and PI3K/Akt pathways, and suggest that EET antagonists are potential therapeutic agents for prostate cancer.

Cerebral cavernous malformations are driven by ADAMTS5 proteolysis of versican.

Cerebral cavernous malformations (CCMs) form following loss of the CCM protein complex in brain endothelial cells due to increased endothelial MEKK3 signaling and KLF2/4 transcription factor expression, but the downstream events that drive lesion formation remain undefined. Recent studies have revealed that CCM lesions expand by incorporating neighboring wild-type endothelial cells, indicative of a cell nonautonomous mechanism. Here we find that endothelial loss of ADAMTS5 reduced CCM formation in the neonatal mouse model. Conversely, endothelial gain of ADAMTS5 conferred early lesion genesis in the absence of increased KLF2/4 expression and synergized with KRIT1 loss of function to create large malformations. Lowering versican expression reduced CCM burden, indicating that versican is the relevant ADAMTS5 substrate and that lesion formation requires proteolysis but not loss of this extracellular matrix protein. These findings identify endothelial secretion of ADAMTS5 and cleavage of versican as downstream mechanisms of CCM pathogenesis and provide a basis for the participation of wild-type endothelial cells in lesion formation.

Permissive microbiome characterizes human subjects with a neurovascular disease cavernous angioma.

Cavernous angiomas (CA) are common vascular anomalies causing brain hemorrhage. Based on mouse studies, roles of gram-negative bacteria and altered intestinal homeostasis have been implicated in CA pathogenesis, and pilot study had suggested potential microbiome differences between non-CA and CA individuals based on 16S rRNA gene sequencing. We here assess microbiome differences in a larger cohort of human subjects with and without CA, and among subjects with different clinical features, and conduct more definitive microbial analyses using metagenomic shotgun sequencing. Relative abundance of distinct bacterial species in CA patients is shown, consistent with postulated permissive microbiome driving CA lesion genesis via lipopolysaccharide signaling, in humans as in mice. Other microbiome differences are related to CA clinical behavior. Weighted combinations of microbiome signatures and plasma inflammatory biomarkers enhance associations with disease severity and hemorrhage. This is the first demonstration of a sensitive and specific diagnostic microbiome in a human neurovascular disease.

Comprehensive transcriptome analysis of cerebral cavernous malformation across multiple species and genotypes.

The purpose of this study was to determine important genes, functions, and networks contributing to the pathobiology of cerebral cavernous malformation (CCM) from transcriptomic analyses across 3 species and 2 disease genotypes. Sequencing of RNA from laser microdissected neurovascular units of 5 human surgically resected CCM lesions, mouse brain microvascular endothelial cells, Caenorhabditis elegans with induced Ccm gene loss, and their respective controls provided differentially expressed genes (DEGs). DEGs from mouse and C. elegans were annotated into human homologous genes. Cross-comparisons of DEGs between species and genotypes, as well as network and gene ontology (GO) enrichment analyses, were performed. Among hundreds of DEGs identified in each model, common genes and 1 GO term (GO:0051656, establishment of organelle localization) were commonly identified across the different species and genotypes. In addition, 24 GO functions were present in 4 of 5 models and were related to cell-to-cell adhesion, neutrophil-mediated immunity, ion transmembrane transporter activity, and responses to oxidative stress. We have provided a comprehensive transcriptome library of CCM disease across species and for the first time to our knowledge in Ccm1/Krit1 versus Ccm3/Pdcd10 genotypes. We have provided examples of how results can be used in hypothesis generation or mechanistic confirmatory studies.