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Neuropilin-1 (NRP1) regulates endothelial cell (EC) biology through modulation of vascular endothelial growth factor receptor 2 (VEGFR2) signalling by presenting VEGFA to VEGFR2. How NRP1 impacts VEGFA-mediated vascular hyperpermeability has however remained unresolved, described as exerting either a positive or a passive function. Using EC-specific Nrp1 knock-out mice, we discover that EC-expressed NRP1 exerts an organotypic role. In the ear skin, VEGFA/VEGFR2-mediated vascular leakage was increased following loss of EC NRP1, implicating NRP1 in negative regulation of VEGFR2 signalling. In contrast, in the back skin and trachea, loss of EC NRP1 decreased vascular leakage. In accordance, phosphorylation of vascular endothelial (VE)-cadherin was increased in the ear skin but suppressed in the back skin of Nrp1 iECKO mice. NRP1 expressed on perivascular cells has been shown to impact VEGF-mediated VEGFR2 signalling. Importantly, expression of NRP1 on perivascular cells was more abundant in the ear skin than in the back skin. Global loss of NRP1 resulted in suppressed VEGFA-induced vascular leakage in the ear skin, implicating perivascular NRP1 as a juxtacrine co-receptor of VEGFA in this compartment. Altogether, we demonstrate that perivascular NRP1 is an active participant in EC VEGFA/VEGFR2 signalling and acts as an organotypic modifier of EC biology.

Idiopathic systemic capillary leak syndrome (ISCLS) is a rare, recurrent condition with dramatically increased blood vessel permeability and, therefore, induction of systemic edema, which may lead to organ damage and death. In this issue of the JCI, Ablooglu et al. showed that ISCLS vessels were hypersensitive to agents known to increase vascular permeability, using human biopsies, cell culture, and mouse models. Several endothelium-specific proteins that regulate endothelial junctions were dysregulated and thereby compromised the vascular barrier. These findings suggest that endothelium-intrinsic dysregulation underlies hyperpermeability and implicate the cytoplasmic serine/threonine protein phosphatase 2A (PP2A) as a potential drug target for the treatment of ISCLS.

TMEM16F (also known as ANO6), a Ca2+-activated 2+-activated lipid scramblase (CaPLSase) that dynamically disrupts lipid asymmetry, plays a crucial role in various physiological and pathological processes, such as blood coagulation, neurodegeneration, cell-cell fusion and viral infection. However, the mechanisms through which it regulates these processes remain largely elusive. Using endothelial cell-mediated angiogenesis as a model, here we report a previously unknown intracellular signaling function of TMEM16F. We demonstrate that TMEM16F deficiency impairs developmental retinal angiogenesis in mice and disrupts angiogenic processes in vitro. Biochemical analyses indicate that the absence of TMEM16F enhances the plasma membrane association of activated Src kinase. This in turn increases VE-cadherin phosphorylation and downregulation, accompanied by suppressed angiogenesis. Our findings not only highlight the role of intracellular signaling by TMEM16F in endothelial cells but also open new avenues for exploring the regulatory mechanisms for membrane lipid asymmetry and their implications in disease pathogenesis.

The endothelial junction component vascular endothelial (VE)–cadherin governs junctional dynamics in the blood and lymphatic vasculature. Here, we explored how lymphatic junction stability is modulated by elevated VEGFA signaling to facilitate metastasis to sentinel lymph nodes. Zippering of VE-cadherin junctions was established in dermal initial lymphatic vessels after VEGFA injection and in tumor-proximal lymphatics in mice. Shape analysis of pan-cellular VE-cadherin fragments revealed that junctional zippering was accompanied by accumulation of small round-shaped VE-cadherin fragments in the lymphatic endothelium. In mice expressing a mutant VEGFR2 lacking the Y949 phosphosite (Vegfr2^Y949F/Y949F) required for activation of Src family kinases, zippering of lymphatic junctions persisted, whereas accumulation of small VE-cadherin fragments was suppressed. Moreover, tumor cell entry into initial lymphatic vessels and subsequent metastatic spread to lymph nodes was reduced in mutant mice compared with WT, after challenge with B16F10 melanoma or EO771 breast cancer. We conclude that VEGFA mediates zippering of VE-cadherin junctions in initial lymphatics. Zippering is accompanied by increased VE-cadherin fragmentation through VEGFA-induced Src kinase activation, correlating with tumor dissemination to sentinel lymph nodes.

Regulation of vascular permeability to plasma is essential for tissue and organ homeostasis and is mediated by endothelial cell-to-cell junctions that tightly regulate the trafficking of molecules between blood and tissue. The single-pass transmembrane glycoprotein CD93 is upregulated in endothelial cells during angiogenesis and controls cytoskeletal dynamics. However, its role in maintaining homeostasis by regulating endothelial barrier function has not been elucidated yet. Here, we demonstrate that CD93 interacts with vascular endothelial (VE)-cadherin and limits its phosphorylation and turnover. CD93 deficiency in vitro and in vivo induces phosphorylation of VE-cadherin under basal conditions, displacing it from endothelial cell–cell contacts. Consistent with this, endothelial junctions are defective in CD93^−/− mice, and the blood–brain barrier permeability is enhanced. Mechanistically, CD93 regulates VE-cadherin phosphorylation and turnover at endothelial junctions through the Rho/Rho kinase-dependent pathway. In conclusion, our results identify CD93 as a key regulator of VE-cadherin stability at endothelial junctions, opening up possibilities for therapeutic strategies directed to control vascular permeability.

Background: Lymphatic vessels are responsible for tissue drainage, and their malfunction is associated with chronic diseases. Lymph uptake occurs via specialized open cell-cell junctions between capillary lymphatic endothelial cells (LECs), whereas closed junctions in collecting LECs prevent lymph leakage. LEC junctions are known to dynamically remodel in development and disease, but how lymphatic permeability is regulated remains poorly understood.

Methods: We used various genetically engineered mouse models in combination with cellular, biochemical, and molecular biology approaches to elucidate the signaling pathways regulating junction morphology and function in lymphatic capillaries.

Results: By studying the permeability of intestinal lacteal capillaries to lipoprotein particles known as chylomicrons, we show that ROCK (Rho-associated kinase)-dependent cytoskeletal contractility is a fundamental mechanism of LEC permeability regulation. We show that chylomicron-derived lipids trigger neonatal lacteal junction opening via ROCK-dependent contraction of junction-anchored stress fibers. LEC-specific ROCK deletion abolished junction opening and plasma lipid uptake. Chylomicrons additionally inhibited VEGF (vascular endothelial growth factor)-A signaling. We show that VEGF-A antagonizes LEC junction opening via VEGFR (VEGF receptor) 2 and VEGFR3-dependent PI3K (phosphatidylinositol 3-kinase)/AKT (protein kinase B) activation of the small GTPase RAC1 (Rac family small GTPase 1), thereby restricting RhoA (Ras homolog family member A)/ROCK-mediated cytoskeleton contraction.

Conclusions: Our results reveal that antagonistic inputs into ROCK-dependent cytoskeleton contractions regulate the interconversion of lymphatic junctions in the intestine and in other tissues, providing a tunable mechanism to control the lymphatic barrier.

Endothelial phospholipase C gamma (PLC gamma) is essential for vascular development; however, its role in healthy, mature, or pathological vessels is unexplored. Here, we show that PLC gamma was prominently expressed in vessels of several human cancer forms, notably in renal cell carcinoma (RCC). High PLC gamma expression in clear cell RCC correlated with angiogenic activity and poor prognosis, while low expression correlated with immune cell activation. PLC gamma was induced downstream of vascular endothelial growth factor receptor 2 (VEGFR2) phosphosite Y1173 (pY1173). Heterozygous Vegfr2Y1173F/+ mice or mice lacking endothelial PLC gamma (Plcg1iECKO) exhibited a stabilized endothelial barrier and diminished vascular leakage. Barrier stabilization was accompanied by decreased expression of immunosuppressive cytokines, reduced infiltration of B cells, helper T cells and regulatory T cells, and improved response to chemo-and immunotherapy. Mechanistically, pY1173/PLC gamma signaling induced Ca2+/protein kinase C-dependent activation of endothelial nitric oxide synthase (eNOS), required for tyrosine nitration and activation of Src. Src-induced phosphorylation of VE-cadherin at Y685 was accompanied by disintegration of endothelial junctions. This pY1173/PLC gamma/eNOS/Src pathway was detected in both healthy and tumor vessels in Vegfr2Y1173F/+ mice, which displayed decreased activation of PLC gamma and eNOS and suppressed vascular leakage. Thus, we believe that we have identified a clinically relevant endothelial PLC gamma pathway downstream of VEGFR2 pY1173, which destabilizes the endothelial barrier and results in loss of antitumor immunity.

A key consequence of increased and sustained vascular permeability in several inflammatory and cardiovascular disorders is the development of interstitial protein-rich proinflammatory edema. This response remains poorly understood mechanistically and its potential adverse effect on local and systemic diseases is often underestimated. To discuss current findings and identify crucial unresolved questions, a workshop was held in Berlin from 12-15 April 2023. Key topics that were discussed included regulation of endothelial cell junctions, neutrophil-dependent vascular leakage, resolution of edema, exemplar diseases, and anti-edema therapies. This report is a summary of the meeting.

Dysfunctional and leaky blood vessels resulting from disruption of the endothelial cell (EC) barrier accompanies numerous diseases. The EC barrier is established through endothelial cell tight and adherens junctions. However, the expression pattern and precise contribution of different junctional proteins to the EC barrier is poorly understood. Here, we focus on organs with continuous endothelium to identify structural and functional in vivo characteristics of the EC barrier. Assembly of multiple single-cell RNAseq datasets into a single integrated database revealed the variability and commonalities of EC barrier patterning. Across tissues, Claudin5 exhibited diminishing expression along the arteriovenous axis, correlating with EC barrier integrity. Functional analysis identified tissue-specific differences in leakage properties and response to the leakage agonist histamine. Loss of Claudin5 enhanced histamine-induced leakage in an organotypic and vessel type-specific manner in an inducible, EC-specific, knock-out mouse. Mechanistically, Claudin5 loss left junction ultrastructure unaffected but altered its composition, with concomitant loss of zonula occludens-1 and upregulation of VE-Cadherin expression. These findings uncover the organ-specific organisation of the EC barrier and distinct importance of Claudin5 in different vascular beds, providing insights to modify EC barrier stability in a targeted, organ-specific manner.

The authors show that Netrin-1-Unc5B signaling controls blood-brain barrier integrity by maintaining Wnt/b-catenin signaling and that delivery of antibodies blocking Netrin-1 binding to Unc5B causes transient and size-selective BBB breakdown. Blood-brain barrier (BBB) integrity is critical for proper function of the central nervous system (CNS). Here, we show that the endothelial Unc5B receptor controls BBB integrity by maintaining Wnt/beta-catenin signaling. Inducible endothelial-specific deletion of Unc5B in adult mice leads to BBB leak from brain capillaries that convert to a barrier-incompetent state with reduced Claudin-5 and increased PLVAP expression. Loss of Unc5B decreases BBB Wnt/beta-catenin signaling, and beta-catenin overexpression rescues Unc5B mutant BBB defects. Mechanistically, the Unc5B ligand Netrin-1 enhances Unc5B interaction with the Wnt co-receptor LRP6, induces its phosphorylation and activates Wnt/beta-catenin downstream signaling. Intravenous delivery of antibodies blocking Netrin-1 binding to Unc5B causes a transient BBB breakdown and disruption of Wnt signaling, followed by neurovascular barrier resealing. These data identify Netrin-1-Unc5B signaling as a ligand-receptor pathway that regulates BBB integrity, with implications for CNS diseases.