Fabrizio Orsenigo

The role of adherens junctions and VE-cadherin in the control of vascular permeability

Endothelial cells control the passage of plasma constituents and circulating cells from blood to the underlying tissues. This specialized function is lost or impaired in several pathological conditions – including inflammation, sepsis, ischemia and diabetes – which leads to severe, and sometimes fatal, organ dysfunction. Endothelial permeability is regulated in part by the dynamic opening and closure of cell-cell adherens junctions (AJs). In endothelial cells, AJs are largely composed of vascular endothelial cadherin (VE-cadherin), an endothelium-specific member of the cadherin family of adhesion proteins that binds, via its cytoplasmic domain, to several protein partners, including p120, β-catenin and plakoglobin. Endogenous pathways that increase vascular permeability affect the function and organization of VE-cadherin and other proteins at AJs in diverse ways. For instance, several factors, including vascular endothelial growth factor (VEGF), induce the tyrosine phosphorylation of VE-cadherin, which accompanies an increase in vascular permeability and leukocyte diapedesis; in addition, the internalization and cleavage of VE-cadherin can cause AJs to be dismantled. From the knowledge of how AJ organization can be modulated, it is possible to formulate several pharmacological strategies to control the barrier function of the endothelium. We discuss the possible use of inhibitors of SRC and other kinases, of agents that increase cAMP levels, and of inhibitors of lytic enzymes as pharmacological tools for decreasing endothelial permeability.

Vascular endothelial cadherin controls VEGFR-2 internalization and signaling from intracellular compartments.

Receptor endocytosis is a fundamental step in controlling the magnitude, duration, and nature of cell signaling events. Confluent endothelial cells are contact inhibited in their growth and respond poorly to the proliferative signals of vascular endothelial growth factor (VEGF). In a previous study, we found that the association of vascular endothelial cadherin (VEC) with VEGF receptor (VEGFR) type 2 contributes to density-dependent growth inhibition (Lampugnani, G.M., A. Zanetti, M. Corada, T. Takahashi, G. Balconi, F. Breviario, F. Orsenigo, A. Cattelino, R. Kemler, T.O. Daniel, and E. Dejana. 2003. J. Cell Biol. 161:793–804). In the present study, we describe the mechanism through which VEC reduces VEGFR-2 signaling. We found that VEGF induces the clathrin-dependent internalization of VEGFR-2. When VEC is absent or not engaged at junctions, VEGFR-2 is internalized more rapidly and remains in endosomal compartments for a longer time. Internalization does not terminate its signaling; instead, the internalized receptor is phosphorylated, codistributes with active phospholipase C–γ, and activates p44/42 mitogen-activated protein kinase phosphorylation and cell proliferation. Inhibition of VEGFR-2 internalization reestablishes the contact inhibition of cell growth, whereas silencing the junction-associated density-enhanced phosphatase-1/CD148 phosphatase restores VEGFR-2 internalization and signaling. Thus, VEC limits cell proliferation by retaining VEGFR-2 at the membrane and preventing its internalization into signaling compartments.

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.

Contact inhibition of VEGF-induced proliferation requires vascular endothelial cadherin, β-catenin, and the phosphatase DEP-1/CD148

Confluent endothelial cells respond poorly to the proliferative signals of VEGF. Comparing isogenic endothelial cells differing for vascular endothelial cadherin (VE-cadherin) expression only, we found that the presence of this protein attenuates VEGF-induced VEGF receptor (VEGFR) 2 phosphorylation in tyrosine, p44/p42 MAP kinase phosphorylation, and cell proliferation. VE-cadherin truncated in β-catenin but not p120 binding domain is unable to associate with VEGFR-2 and to induce its inactivation. β-Catenin–null endothelial cells are not contact inhibited by VE-cadherin and are still responsive to VEGF, indicating that this protein is required to restrain growth factor signaling. A dominant-negative mutant of high cell density–enhanced PTP 1 (DEP-1)//CD148 as well as reduction of its expression by RNA interference partially restore VEGFR-2 phosphorylation and MAP kinase activation. Overall the data indicate that VE-cadherin–β-catenin complex participates in contact inhibition of VEGF signaling. Upon stimulation with VEGF, VEGFR-2 associates with the complex and concentrates at cell–cell contacts, where it may be inactivated by junctional phosphatases such as DEP-1. In sparse cells or in VE-cadherin–null cells, this phenomenon cannot occur and the receptor is fully activated by the growth factor.

Interaction of Junctional Adhesion Molecule with the Tight Junction Components ZO-1, Cingulin, and Occludin

Junctional adhesion molecule (JAM) is an integral membrane protein that has been reported to colocalize with the tight junction molecules occludin, ZO-1, and cingulin. However, evidence for the association of JAM with these molecules is missing. Transfection of Chinese hamster ovary cells with JAM (either alone or in combination with occludin) resulted in enhanced junctional localization of both endogenous ZO-1 and cotransfected occludin. Additionally, JAM was coprecipitated with ZO-1 in the detergent-insoluble fraction of Caco-2 epithelial cells. A putative PDZ-binding motif at the cytoplasmic carboxyl terminus of JAM was required for mediating the interaction of JAM with ZO-1, as assessed by in vitro binding and coprecipitation experiments. JAM was also coprecipitated with cingulin, another cytoplasmic component of tight junctions, and this association required the amino-terminal globular head of cingulin. Taken together, these data indicate that JAM is a component of the multiprotein complex of tight junctions, which may facilitate junction assembly.

Sox18 induces development of the lymphatic vasculature in mice

The lymphatic system plays a key role in tissue fluid regulation and tumour metastasis, and lymphatic defects underlie many pathological states including lymphoedema, lymphangiectasia, lymphangioma and lymphatic dysplasia. However, the origins of the lymphatic system in the embryo, and the mechanisms that direct growth of the network of lymphatic vessels, remain unclear. Lymphatic vessels are thought to arise from endothelial precursor cells budding from the cardinal vein under the influence of the lymphatic hallmark gene Prox1 (prospero homeobox 1; ref. 4). Defects in the transcription factor gene SOX18 (SRY (sex determining region Y) box 18) cause lymphatic dysfunction in the human syndrome hypotrichosis-lymphoedema-telangiectasia, suggesting that Sox18 may also play a role in lymphatic development or function. Here we use molecular, cellular and genetic assays in mice to show that Sox18 acts as a molecular switch to induce differentiation of lymphatic endothelial cells. Sox18 is expressed in a subset of cardinal vein cells that later co-express Prox1 and migrate to form lymphatic vessels. Sox18 directly activates Prox1 transcription by binding to its proximal promoter. Overexpression of Sox18 in blood vascular endothelial cells induces them to express Prox1 and other lymphatic endothelial markers, while Sox18-null embryos show a complete blockade of lymphatic endothelial cell differentiation from the cardinal vein. Our findings demonstrate a critical role for Sox18 in developmental lymphangiogenesis, and suggest new avenues to investigate for therapeutic management of human lymphangiopathies.

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.

CCM1 regulates vascular-lumen organization by inducing endothelial polarity

Little is known about the molecular mechanisms that regulate the organization of vascular lumen. In this paper we show that lumen formation correlates with endothelial polarization. Adherens junctions (AJs) and VE-cadherin (VEC, encoded by CDH5) are required for endothelial apicobasal polarity in vitro and during embryonic development. Silencing of CDH5 gene expression leads to abrogation of endothelial polarity accompanied by strong alterations in lumenal structure. VEC co-distributes with members of the Par polarity complex (Par3 and PKCζ) and is needed for activation of PKCζ. CCM1 is encoded by the CCM1 gene, which is mutated in 60% of patients affected by cerebral cavernous malformation (CCM). The protein interacts with VEC and directs AJ organization and AJ association with the polarity complex, both in cell-culture models and in human CCM1 lesions. Both VEC and CCM1 control Rap1 concentration at cell-cell junctions. We propose that VEC, CCM1 and Rap1 form a signaling complex. In the absence of any of these proteins, AJs are dismantled, cell polarity is lost and vascular lumenal structure is severely altered.