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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 Ca 2+ /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 [Ca 2+ ] 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 β-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 Ca 2+ homeostasis and barrier function in human retinal capillaries and suggest that TRPV4 may differentially contribute to the inner vs. outer blood-retinal barrier function., (© 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.)

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/β-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.

The vascular endothelium responds to infection by destabilizing endothelial cell-cell junctions to allow fluid and cells to pass into peripheral tissues, facilitating clearance of infection and tissue repair. During sepsis, endotoxin and other proinflammatory molecules induce excessive vascular leak, which can cause organ dysfunction, shock, and death. Current therapies for sepsis are limited to antibiotics and supportive care, which are often insufficient to reduce morbidity and prevent mortality. Previous attempts at blocking inflammatory cytokine responses in humans proved ineffective at reducing the pathologies associated with sepsis, highlighting the need for a new therapeutic strategy. The small GTPase ARF6 is activated by a MyD88-ARNO interaction to induce vascular leak through disruption of endothelial adherens junctions. In this study, we show that the MyD88-ARNO-ARF6-signaling axis is responsible for LPS-induced endothelial permeability and is a destabilizing convergence point used by multiple inflammatory cues. We also show that blocking ARF6 with a peptide construct of its N terminus is sufficient to reduce vascular leak and enhance survival during endotoxic shock, without inhibiting the host cytokine response. Our data highlight the therapeutic potential of blocking ARF6 and reducing vascular leak for the treatment of inflammatory conditions, such as endotoxemia., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Rationale: Netrin-4 regulates vascular development. Identity of netrin-4 endothelial receptor and its subsequent cell functions is controversial. We previously demonstrated that the inhibition of netrin-1 canonical receptors, Unc5B and neogenin, expressed by lymphatic endothelial cells, do not suppress netrin-4-induced cell signaling and functions. Netrin family members were shown to signal through a range of receptors, including integrins (such as α3β1, α6β1, and α6β4) in nonendothelial cells., Objective: We tested whether integrins are netrin-4 receptors in the endothelium., Methods and Results: The α6β1 integrin is expressed by endothelial cells, and binds netrin-4 in a dose-dependent manner. Inhibition of α6 or β1 integrin subunits suppresses netrin-4-induced endothelial cell migration, adhesion, and focal adhesion contact. Netrin-4-stimulated phosphorylation of Src kinase family, effectors of endothelial cell migration, is also abolished by α6 or β1 inhibition. Finally, netrin-4 and α6β1 integrin expression colocalize in mouse embryonic, intestine, and tumor vasculature., Conclusions: The α6β1 integrin is a netrin-4 receptor in lymphatic endothelium and consequently represents a potential target to inhibit netrin-4-induced metastatic dissemination.

Elastin is an essential component of arteries which provides structural integrity and instructs smooth muscle cells to adopt a quiescent state. Despite interaction of endothelial cells with elastin in the internal elastic lamina, the potential for exploiting this interaction therapeutically has not been explored in detail. In this study, we show that tropoelastin (a precursor of elastin) stimulates endothelial cell migration and adhesion more than smooth muscle cells. The biological activity of tropoelastin on endothelial cells is contained in the VGVAPG domain and in the carboxy-terminal 17-amino acids. We show that the effects of the carboxy-terminal 17 amino acids, but not those of VGVAPG, are mediated by integrin α(V)β(3). We demonstrate that tropoelastin covalently linked to stainless steel disks promotes adhesion of endothelial progenitor cells and endothelial cells to the metal surfaces. The adherent cells on the tropoelastin-coated metal surfaces form monolayers that can withstand and respond to arterial shear stress. Because of the unique effects of tropoelastin on endothelial and smooth muscle cells, coating intravascular devices with tropoelastin may stimulate their endothelialization, inhibit smooth muscle hyperplasia, and improve device performance.

Netrin-4, a laminin-related secreted protein is an axon guidance cue recently shown essential outside of the nervous system, regulating mammary and lung morphogenesis as well as blood vascular development. Here, we show that Netrin-4, at physiologic doses, induces proliferation, migration, adhesion, tube formation and survival of human lymphatic endothelial cells in vitro comparable to well-characterized lymphangiogenic factors fibroblast growth factor-2 (FGF-2), hepatocyte growth factor (HGF), vascular endothelial growth factor-A (VEGF-A), and vascular endothelial growth factor-C (VEGF-C). Netrin-4 stimulates phosphorylation of intracellular signaling components Akt, Erk and S6, and their specific inhibition antagonizes Netrin-4-induced proliferation. Although Netrin receptors Unc5B and neogenin, are expressed by human lymphatic endothelial cells, suppression of either or both does not suppress Netrin-4-promoted in vitro effects. In vivo, Netrin-4 induces growth of lymphatic and blood vessels in the skin of transgenic mice and in breast tumors. Its overexpression in human and mouse mammary carcinoma cancer cells leads to enhanced metastasis. Finally, Netrin-4 stimulates in vitro and in vivo lymphatic permeability by activating small GTPases and Src family kinases/FAK, and down-regulating tight junction proteins. Together, these data provide evidence that Netrin-4 is a lymphangiogenic factor contributing to tumor dissemination and represents a potential target to inhibit metastasis formation.

The function of the endothelium is to provide a network to allow delivery of oxygen and nutrients to tissues throughout the body. This network comprises adjacent endothelial cells that utilize adherens junction proteins such as vascular endothelial cadherin (VE-cadherin) to maintain the appropriate level of vascular permeability. The disruption of VE-cadherin interactions during pathologic settings can lead to excessive vascular leak with adverse effects. Endogenous cell signaling systems have been defined, which help to maintain the proper level of vascular stability. Perhaps the best described system is Angiopoietin-1 (Ang-1). Ang-1 acting through its receptor Tie2 generates a well-described set of signaling events ultimately leading to enhanced vascular stability. In this review, we will focus on what is known about additional endogenous cell signaling systems that stabilize the vasculature, and using Ang-1/Tie2 as a model, we will address where our understanding of these additional systems is lacking.

Axonal guidance and vascular patterning share several guidance cues, including proteins in the netrin family. We demonstrate that netrins stimulate proliferation, migration, and tube formation of human endothelial cells in vitro and that this stimulation is independent of known netrin receptors. Suppression of netrin1a messenger RNA in zebrafish inhibits vascular sprouting, implying a proangiogenic role for netrins during vertebrate development. We also show that netrins accelerate neovascularization in an in vivo model of ischemia and that they reverse neuropathy and vasculopathy in a diabetic murine model. We propose that the attractive vascular and neural guidance functions of netrins offer a unique therapeutic potential.

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.

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. [ABSTRACT FROM AUTHOR]