Costanza Giampietro

Endothelial adherens junctions control tight junctions by VE-cadherin-mediated upregulation of claudin-5.

Intercellular junctions mediate adhesion and communication between adjoining cells. Although formed by different molecules, tight junctions (TJs) and adherens junctions (AJs) are functionally and structurally linked, but the signalling pathways behind this interaction are unknown. Here we describe a cell-specific mechanism of crosstalk between these two types of structure. We show that endothelial VE-cadherin at AJs upregulates the gene encoding the TJ adhesive protein claudin-5. This effect requires the release of the inhibitory activity of forkhead box factor FoxO1 and the Tcf-4–β-catenin transcriptional repressor complex. Vascular endothelial (VE)-cadherin acts by inducing the phosphorylation of FoxO1 through Akt activation and by limiting the translocation of β-catenin to the nucleus. These results offer a molecular basis for the link between AJs and TJs and explain why VE-cadherin inhibition may cause a marked increase in permeability.

Phosphorylation of VE-cadherin is modulated by haemodynamic forces and contributes to the regulation of vascular permeability in vivo

Endothelial adherens junctions maintain vascular integrity. Arteries and veins differ in their permeability but whether organization and strength of their adherens junctions vary has not been demonstrated in vivo. Here we report that vascular endothelial cadherin, an endothelial specific adhesion protein located at adherens junctions, is phosphorylated in Y658 and Y685 in vivo in veins but not in arteries under resting conditions. This difference is due to shear stress-induced junctional Src activation in veins. Phosphorylated vascular endothelial-cadherin is internalized and ubiquitinated in response to permeability-increasing agents such as bradykinin and histamine. Inhibition of Src blocks vascular endothelial cadherin phosphorylation and bradykinin-induced permeability. Point mutation of Y658F and Y685F prevents vascular endothelial cadherin internalization, ubiquitination and an increase in permeability by bradykinin in vitro. Thus, phosphorylation of vascular endothelial cadherin contributes to a dynamic state of adherens junctions, but is not sufficient to increase vascular permeability in the absence of inflammatory agents.

EndMT contributes to the onset and progression of cerebral cavernous malformations

Cerebral cavernous malformation (CCM) is a vascular dysplasia, mainly localized within the brain and affecting up to 0.5% of the human population. CCM lesions are formed by enlarged and irregular blood vessels that often result in cerebral haemorrhages. CCM is caused by loss-of-function mutations in one of three genes, namely CCM1 (also known as KRIT1), CCM2 (OSM) and CCM3 (PDCD10), and occurs in both sporadic and familial forms. Recent studies have investigated the cause of vascular dysplasia and fragility in CCM, but the in vivo functions of this ternary complex remain unclear. Postnatal deletion of any of the three Ccm genes in mouse endothelium results in a severe phenotype, characterized by multiple brain vascular malformations that are markedly similar to human CCM lesions. Endothelial-to-mesenchymal transition (EndMT) has been described in different pathologies, and it is defined as the acquisition of mesenchymal- and stem-cell-like characteristics by the endothelium. Here we show that endothelial-specific disruption of the Ccm1 gene in mice induces EndMT, which contributes to the development of vascular malformations. EndMT in CCM1-ablated endothelial cells is mediated by the upregulation of endogenous BMP6 that, in turn, activates the transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP) signalling pathway. Inhibitors of the TGF-β and BMP pathway prevent EndMT both in vitro and in vivo and reduce the number and size of vascular lesions in CCM1-deficient mice. Thus, increased TGF-β and BMP signalling, and the consequent EndMT of CCM1-null endothelial cells, are crucial events in the onset and progression of CCM disease. These studies offer novel therapeutic opportunities for this severe, and so far incurable, pathology.

The Wnt/β-Catenin Pathway Modulates Vascular Remodeling and Specification by Upregulating Dll4/Notch Signaling

The Wnt/beta-catenin pathway is evolutionary conserved signaling system that regulates cell differentiation and organogenesis. We show that endothelial specific stabilization of Wnt/beta-catenin signaling alters early vascular development in the embryo. The phenotype resembles that induced by upregulation of Notch signaling, including lack of vascular remodeling, altered elongation of the intersomitic vessels, defects in branching, and loss of venous identity. Both in vivo and in vitro data show that beta-catenin upregulates Dll4 transcription and strongly increases Notch signaling in the endothelium, leading to functional and morphological alterations. The functional consequences of beta-catenin signaling depend on the stage of vascular development and are lost when a gain-of-function mutation is induced at a late stage of development or postnatally. Our findings establish a link between Wnt and Notch signaling in vascular development. We propose that early and sustained beta-catenin signaling prevents correct endothelial cell differentiation, altering vascular remodeling and arteriovenous specification.

Vascular endothelial-cadherin and vascular stability.

Purpose of reviewVascular integrity is characterized by a tight control of permeability to cells and solutes and by resistance to blood flow. In several pathologies including tumor angiogenesis, vascular malformations, hemorrhagic stroke and others, there is the need to stabilize the vessels and prevent undesired bleeding or edema. Here, we discuss the current knowledge on the role of endothelial cell-to-cell adherens junctions in maintaining vascular integrity. Recent findingsThe identification of several components of adherens junctions in endothelial cells helped understanding of the complex role of these structures not only in maintaining cell-to-cell adhesion but also in transferring intracellular signals. Vascular endothelial (VE)-cadherin, an endothelial-specific adhesion protein at adherens junctions, was found to interact with several signaling partners that induce contact inhibition of growth, decrease in permeability, tight junction organization and others. Changes in VE-cadherin levels in vivo may significantly affect vascular permeability, and induce uncontrolled growth and vascular fragility. SummaryIn the past years, the research on angiogenesis was mostly directed to the definition of the mechanisms able to modulate vascular growth. We now understand that in many pathological conditions we do not simply need to increase or inhibit vascularization but we also need to develop tools able to stabilize organ perfusion and to avoid hemorrhages or edema.

VE‐cadherin is a critical endothelial regulator of TGF‐β signalling

VE‐cadherin is an endothelial‐specific transmembrane protein concentrated at cell‐to‐cell adherens junctions. Besides promoting cell adhesion and controlling vascular permeability, VE‐cadherin transfers intracellular signals that contribute to vascular stabilization. However, the molecular mechanism by which VE‐cadherin regulates vascular homoeostasis is still poorly understood. Here, we report that VE‐cadherin expression and junctional clustering are required for optimal transforming growth factor‐β (TGF‐β) signalling in endothelial cells (ECs). TGF‐β antiproliferative and antimigratory responses are increased in the presence of VE‐cadherin. ECs lacking VE‐cadherin are less responsive to TGF‐β/ALK1‐ and TGF‐β/ALK5‐induced Smad phosphorylation and target gene transcription. VE‐cadherin coimmunoprecipitates with all the components of the TGF‐β receptor complex, TβRII, ALK1, ALK5 and endoglin. Clustered VE‐cadherin recruits TβRII and may promote TGF‐β signalling by enhancing TβRII/TβRI assembly into an active receptor complex. Taken together, our data indicate that VE‐cadherin is a positive and EC‐specific regulator of TGF‐β signalling. This suggests that reduction or inactivation of VE‐cadherin may contribute to progression of diseases where TGF‐β signalling is impaired.

Overlapping and divergent signaling pathways of N-cadherin and VE-cadherin in endothelial cells

Endothelial cells (ECs) express 2 members of the cadherin family, VE and N-cadherin. Although VE-cadherin induces EC homotypic adhesion, N-cadherin function in ECs remains largely unknown. EC-specific inactivation of either VE or N-cadherin leads to early fetal lethality suggesting that these cadherins play a nonredundant role in vascular development. We report here that VE-cadherin negatively controls junctional localization and expression of N-cadherin by limiting p120-catenin availability and reducing β-catenin transcriptional activity. Using EC lines expressing either VE or N-cadherin we found that both cadherins inhibit cell proliferation and apoptosis. Both trigger the phosphatidylinositol-3-OH-kinase (PI3K)-AKT-Forkhead-box protein-O1 (FoxO1) pathway and reduce β-catenin transcriptional activity. The extent of signaling correlates with the total level of cadherins regardless of the type of cadherin expressed. In contrast, basal and fibroblast growth factor (FGF)-induced cell motility is promoted by N-cadherin and strongly inhibited by VE-cadherin. This opposite effect is partly because of the ability of VE-cadherin to associate with FGF receptor and the density-enhanced phosphatase-1 (Dep-1) which, in turn, inhibits receptor signaling. We conclude that VE and N-cadherin have both additive and divergent effects on ECs. Differences in signaling are due, in part, to cadherin association with growth factor receptors and modulation of their downstream signaling.

KLF4 is a key determinant in the development and progression of cerebral cavernous malformations

Cerebral cavernous malformations (CCMs) are vascular malformations located within the central nervous system often resulting in cerebral hemorrhage. Pharmacological treatment is needed, since current therapy is limited to neurosurgery. Familial CCM is caused by loss‐of‐function mutations in any of Ccm1, Ccm2, and Ccm3 genes. CCM cavernomas are lined by endothelial cells (ECs) undergoing endothelial‐to‐mesenchymal transition (EndMT). This switch in phenotype is due to the activation of the transforming growth factor beta/bone morphogenetic protein (TGFβ/BMP) signaling. However, the mechanism linking Ccm gene inactivation and TGFβ/BMP‐dependent EndMT remains undefined. Here, we report that Ccm1 ablation leads to the activation of a MEKK3‐MEK5‐ERK5‐MEF2 signaling axis that induces a strong increase in Kruppel‐like factor 4 (KLF4) in ECs in vivo. KLF4 transcriptional activity is responsible for the EndMT occurring in CCM1‐null ECs. KLF4 promotes TGFβ/BMP signaling through the production of BMP6. Importantly, in endothelial‐specific Ccm1 and Klf4 double knockout mice, we observe a strong reduction in the development of CCM and mouse mortality. Our data unveil KLF4 as a therapeutic target for CCM.

Sulindac metabolites decrease cerebrovascular malformations in CCM3-knockout mice

Significance Cerebral cavernous malformation (CCM) disease can lead to brain hemorrhages, seizures, and paralysis. No pharmacological therapy is currently available. Here we define, to our knowledge for the first time in vivo, the sequence of molecular events that lead to CCM vascular cavernomas. We found that β-catenin activation is the first trigger followed by TGF-β signaling, which, in turn, mediates the progression of the disease. We also show that β-catenin signaling is cell-autonomous and independent of Wnt-receptor activation. Most importantly, these studies prompted us to identify pharmacological agents that, by targeting the altered β-catenin signaling, limit the formation of brain vascular cavernomas in mice with CCM3 ablation in endothelial cells. These drugs are currently used in clinics for different pathologies and may be repurposed for CCM therapy. Cerebral cavernous malformation (CCM) is a disease of the central nervous system causing hemorrhage-prone multiple lumen vascular malformations and very severe neurological consequences. At present, the only recommended treatment of CCM is surgical. Because surgery is often not applicable, pharmacological treatment would be highly desirable. We describe here a murine model of the disease that develops after endothelial-cell–selective ablation of the CCM3 gene. We report an early, cell-autonomous, Wnt-receptor–independent stimulation of β-catenin transcription activity in CCM3-deficient endothelial cells both in vitro and in vivo and a triggering of a β-catenin–driven transcription program that leads to endothelial-to-mesenchymal transition. TGF-β/BMP signaling is then required for the progression of the disease. We also found that the anti-inflammatory drugs sulindac sulfide and sulindac sulfone, which attenuate β-catenin transcription activity, reduce vascular malformations in endothelial CCM3-deficient mice. This study opens previously unidentified perspectives for an effective pharmacological therapy of intracranial vascular cavernomas.

Deciphering the functional role of endothelial junctions by using in vivo models

Endothelial cell‐to‐cell junctions are vital for the formation and integrity of blood vessels. The main adhesive junctional complexes in endothelial cells, adherens junctions and tight junctions, are formed by transmembrane adhesive proteins that are linked to intracellular signalling partners and cytoskeletal‐binding proteins. Gene inactivation and blocking antibodies in mouse models have revealed some of the functions of the individual junctional components in vivo, and are increasing our understanding of the functional role of endothelial cell junctions in angiogenesis and vascular homeostasis. Adherens‐junction organization is required for correct vascular morphogenesis during embryo development. By contrast, the data available suggest that tight‐junction proteins are not essential for vascular development but are necessary for endothelial barrier function.