Endothelial Cell Adherens Junctions Research Articles

Nuciferine reduces vascular leakage and improves cardiac function in acute myocardial infarction by regulating the PI3K/AKT pathway

The destruction of the microvascular structure and function can seriously affect the survival and prognosis of patients with acute myocardial infarction (AMI). Nuciferine has a potentially beneficial effect in the treatment of cardiovascular disease, albeit its role in microvascular structure and function during AMI remains unclear. This study aimed to investigate the protective effect and the related mechanisms of nuciferine in microvascular injury during AMI. Cardiac functions and pathological examination were conducted in vivo to investigate the effect of nuciferine on AMI. The effect of nuciferine on permeability and adherens junctions in endothelial cells was evaluated in vitro, and the phosphorylation level of the PI3K/AKT pathway (in the presence or absence of PI3K inhibitors) was also analyzed. In vivo results indicated that nuciferine inhibited ischemia-induced cardiomyocyte damage and vascular leakage and improved cardiac function. In addition, the in vitro results revealed that nuciferine could effectively inhibit oxygen–glucose deprivation (OGD) stimulated breakdown of the structure and function of human coronary microvascular endothelial cells (HCMECs). Moreover, nuciferine could significantly increase the phosphorylation level of the PI3K/AKT pathway. Finally, the inhibitor wortmannin could reverse the protective effect of nuciferine on HCMECs. Nuciferine inhibited AMI-induced microvascular injury by regulating the PI3K/AKT pathway and protecting the endothelial barrier function in mice.

Rictor maintains endothelial integrity under shear stress

Background: Endothelial injury induced by low shear stress (LSS) is an initiating factor in the pathogenesis of various cardiovascular diseases, including atherosclerosis, hypertension, and thrombotic diseases. Low shear stress activates the mammalian target of rapamycin complex 2 (mTORC2) signaling pathway. Rictor, the main constituent protein of mTORC2, is involved in vascular development. However, the impact of conditional Rictor ablation on endothelial homeostasis, especially on endothelial-specific markers, such as vascular endothelial-cadherin (VE-cadherin) and von Willebrand factor (VWF), under blood flow stimulation is unclear. Objective: We aimed to investigate whether endothelial Rictor is involved in maintaining vascular endothelial integrity and the potential role of Rictor in atheroprone blood flow-mediated endothelial injury. Methods and results: Immunofluorescence staining showed that endothelial Rictor was successfully knocked out in a mouse model. Scanning electron microscopy (EM) detection revealed disruption of the endothelial monolayer in the thoracic aorta of Rictor-deficient mice. Furthermore, scanning electron microscopy and transmission electron microscopy showed that Rictor deletion disrupted endothelial integrity and expanded cell junctions in the left common carotid artery region. In vitro, low shear stress disrupted actin filament polarity and the promoted the translocation of vascular endothelial-cadherin, the key component of adherens junctions (AJs) in human umbilical vein endothelial cells. After Rictor downregulation by small interfering RNA, the translocation of vascular endothelial-cadherin and stress fibers increased. Rictor knockdown inhibited low shear stress-induced von Willebrand factor upregulation, and downregulation of vascular endothelial-cadherin decreased low shear stress-induced von Willebrand factor expression. These results suggest that vascular endothelial-cadherin/von Willebrand factor is a possible mechanism mediated by Rictor in the pathological process of low shear stress-induced endothelial injury. Conclusion: Rictor is a key protein that regulates endothelial integrity under vascular physiological homeostasis, and Rictor mediates low shear stress-induced endothelial injury by regulating adherens junctions and von Willebrand factor.

Claudin5 protects the peripheral endothelial barrier in an organ and vessel-type-specific manner.

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.

Induction of endothelial barrier dysfunction by serum factors in rats subjected to traumatic brain injury and hemorrhagic shock.

Traumatic brain injury (TBI) has been associated with the development of indirect acute respiratory distress syndrome (ARDS). However, the causative relationship between TBI and lung injury remains unclear. To explore potential mechanisms linking TBI with the development of ARDS, we characterized the effects of serum factors released following TBI and hemorrhagic shock (HS) in a rat model on the pulmonary endothelial cell (EC) barrier dysfunction, a key feature of ARDS. We found that serum samples from animals exposed to both controlled cortical impact (CCI) and HS, but not from sham‐operated rats induced significant barrier dysfunction in human pulmonary artery EC monolayers at 2 days post injury. Thrombin inhibitor and thrombin receptor antagonist attenuated the acute phase of the serum‐induced trans‐endothelial resistance (TER) decline caused by CCI‐HS serum, but not in later time points. However, both the early and late phases of CCI‐HS‐induced EC permeability were inhibited by heparin. The barrier disruptive effects of CCI‐HS serum were also prevented by serum preincubation with heparin‐sepharose. Pulmonary EC treated for 3 h with serum from CCI‐HS rats demonstrated a significant decline in expression of EC junctional protein, VE‐Cadherin, and disassembly of peripheral EC adherens junction complexes monitored by immunostaining with VE‐cadherin antibody. These results suggest that exposure to CCI‐HS causes early and late‐phase barrier disruptive effects in vascular endothelium. While thrombin‐PAR1 signaling has been identified as a mechanism of acute EC permeability increase by CCI‐HS serum, the factor(s) defining long‐term EC barrier disruption in CCI‐HS model remains to be determined.

Serum from rats subjected to traumatic brain injury and hemorrhagic shock induces profound endothelial barrier dysfunction

IntroductionTraumatic brain injury (TBI) has been associated with development of indirect acute respiratory distress syndrome (ARDS). However, the causative relationship between TBI and lung injury remains unclear. To explore potential mechanism linking TBI with development of ARDS, we characterized effects of serum factors released during TBI and hemorrhagic shock (HS) in a rat model on the pulmonary endothelial cell (EC) barrier dysfunction, a key feature of ARDS.MethodsThe adult male rat polytrauma model consisted of controlled cortical impact (CCI)–induced TBI followed by 30 minutes of hemorrhage shock (HS) induced by blood withdrawal. The HS phase was followed by a 1‐hour “prehospital” Hextend fluid resuscitation phase and then a 1‐hour “hospital phase” when shed blood was reinfused. 48 hours after the TBI, blood was drawn from right ventricle and serum samples were obtained. Serum was collected from the TBI or sham rats, and its effects on barrier properties of pulmonary endothelial cell culture were assessed by measurements of transendothelial electrical resistance (TER) using the electrical cell impedance sensor and visualization of fluorescent tracer penetration through EC monolayer using XPerT assay.ResultsWe found pronounced barrier‐disruptive effects of serum samples from animals exposed to controlled cortical impact (CCI) and HS, but not from sham operated rats. Rapid and sustained decrease in EC monolayer transendothelial resistance was observed within minutes after serum addition and remained below basal TER levels after incubation with CCI‐HS sera. Thrombin has been implicated in the early‐phase barrier disruptive activity of CCI‐HS serum, as both thrombin inhibitor and thrombin receptor antagonist attenuated the acute phase of serum‐induced TER decline, but did not affect TER decline at later time points. These findings suggest that thrombin elevation may play a role in early‐phase barrier disruptive activity of CCI‐HS serum. However, both the early and late phases of CCI‐HS‐induced EC permeability were inhibited by heparin. The late phase barrier disruptive effect of CCI‐HS was also prevented by serum preincubation with heparin‐sepharose. Pulmonary EC treated with serum from CCI‐HS rats for 3 hours have demonstrated significant decline in expression of EC junctional protein, VE‐Cadherin, and disassembly of peripheral EC adherens junction complexes monitored by immunostaining with VE‐cadherin antibody.ConclusionThese results suggest that exposure to CCI‐HS leads to production of humoral factors causing early and late‐phase barrier disruptive effects in lung vascular endothelium. While thrombin‐PAR1 signaling has been identified as mechanism of acute EC permeability increase by CCI‐HS serum, the factor(s) defining long‐term EC barrier disruption in CCI‐HS model remain to be determined.

ETS-Related Gene Activation Preserves Adherens Junctions and Permeability in Microvascular Endothelial Cells.

ERG (ETS-related gene) is a member of the ETS (Erythroblast-transformation specific) family of transcription factors abundantly present in vascular endothelial cells. Recent studies demonstrate that ERG has important roles in blood vessel stability and angiogenesis. However, it is unclear how ERG is potentially involved in microvascular barrier functions and permeability. A wide variety of diseases and clinical conditions including trauma-hemorrhagic shock and burn injury are associated with microvascular dysfunctions, which causes excessive microvascular permeability, tissue edema and eventually, multiple organ dysfunction and death. The main purpose of this study was to determine the specific role of ERG in regulating microvascular permeability in human lung microvascular endothelial cells (HLMEC) and to evaluate if exogenous ERG will protect the barrier. The HLMECs were grown on Transwell inserts as monolayers and were transfected with ERG CRISPR/cas9 knockdown plasmid, ERG CRISPR activation plasmid, recombinant ERG protein or their respective controls. Recombinant vascular endothelial growth factor (VEGF) was used as an inducer of permeability for evaluating the effect of ERG activation on permeability. Changes in barrier integrity and permeability were studied using monolayer permeability assay and immunofluorescence of adherens junction proteins (VE-cadherin and β-catenin) respectively. CRISPR/cas9-based ERG knockdown as well as VEGF treatment induced monolayer hyperpermeability, VE-cadherin, and β-catenin junctional relocation and cytoskeletal F-actin stress fiber formation. CRISPR based ERG activation and recombinant ERG transfection attenuated VEGF-induced monolayer hyperpermeability. ERG activation preserved the adherens junctions and cytoskeleton. These results demonstrate that ERG is a potent regulator of barrier integrity and permeability in human lung microvascular endothelial cells and endogenously or exogenously enhancing ERG provides protection against barrier dysfunction and hyperpermeability.

Nectin stabilization at adherens junctions is counteracted by Rab5a-dependent endocytosis.

Cell-cell junctions undergo constant remodeling, which is crucial for the control of vascular integrity. Indeed, transport of junctional components such as cadherins is understood in increasing depth. However, little is known about the respective pathways regulating localization of nectin at cell-cell junctions. Here, we performed an siRNA-based screen of vesicle regulators of the RabGTPase family, leading to the identification of a novel role for Rab5a in the endocytosis nectin-2 at adherens junctions of primary human endothelial cells (HUVEC). Confocal microscopy experiments revealed disordered nectin-2 localization at adherens junctions upon Rab5a depletion. In addition, internalized nectin-2 was shown to prominently localize to Rab5a-positive vesicles in both fixed and living cells. As shown previously, nectin-2 stabilization at junctions is achieved via drebrin-dependent coupling to the subcortical actin cytoskeleton. Consistently, depletion of drebrin in this study leads to enhanced internalization of nectin-2 from junctions. Strikingly, simultaneous silencing of Rab5a and drebrin restored the junctional localization of nectin-2, pointing to Rab5a as counteracting the drebrin-dependent stabilization of nectin-2 at adherens junctions. This mechanism could be further validated by transendothelial resistance measurements. Collectively, our results identify Rab5a as a key player in the endocytosis of nectin-2 and thus in the regulation of adherens junction integrity in primary human endothelial cells.

RhoGEF17-An Essential Regulator of Endothelial Cell Death and Growth.

The Rho guanine nucleotide exchange factor RhoGEF17 was described to reside in adherens junctions (AJ) in endothelial cells (EC) and to play a critical role in the regulation of cell adhesion and barrier function. The purpose of this study was to analyze signal cascades and processes occurring subsequent to AJ disruption induced by RhoGEF17 knockdown. Primary human and immortalized rat EC were used to demonstrate that an adenoviral-mediated knockdown of RhoGEF17 resulted in cell rounding and an impairment in spheroid formation due to an enhanced proteasomal degradation of AJ components. In contrast, β-catenin degradation was impaired, which resulted in an induction of the β-catenin-target genes cyclin D1 and survivin. RhoGEF17 depletion additionally inhibited cell adhesion and sheet migration. The RhoGEF17 knockdown prevented the cells with impeded cell–cell and cell–matrix contacts from apoptosis, which was in line with a reduction in pro-caspase 3 expression and an increase in Akt phosphorylation. Nevertheless, the cells were not able to proliferate as a cell cycle block occurred. In summary, we demonstrate that a loss of RhoGEF17 disturbs cell–cell and cell–substrate interaction in EC. Moreover, it prevents the EC from cell death and blocks cell proliferation. Non-canonical β-catenin signaling and Akt activation could be identified as a potential mechanism.

Hydrodynamic forces influence the gene transcription of mechanosensitive intercellular junction associated genes in corneal endothelial cells

Mechanicals forces are known to influence cell behavior. In vivo, the corneal endothelium is under the influence of various mechanical forces, such as intraocular pressure (IOP) and fluid flow. In this study, we used a corneal bioreactor to understand the effect of these hydrodynamic forces on the transcription of intercellular junctions associated genes in the corneal endothelium. Native and tissue-engineered (TE) corneal endothelium were cultured in a corneal bioreactor for 7 days with 16 mmHg IOP and 5 μl/ml of medium flow. RNA was harvested, and gene expression was quantified. Cells that were used to reconstruct the TE corneal endothelia were also seeded on plastic to characterize their morphology by calculating their circularity index. For native endothelia, hydrodynamic forces increased gene expression of GJA1 (connexin 43), CDH2 (N-cadherin), TJP1 (ZO-1), ITGAV (integrin subunit αv), ITGB5 (integrin subunit β5) and CTNND1 (p120-ctn) by 1.68 ± 0.40, 1.10 ± 0.27, 3.80 ± 0.56, 1.82 ± 0.33, 1.32 ± 0.21 and 3.04 ± 0.63, respectively. For TE corneal endothelium, this fold change was 1.72 ± 0.31, 1.58 ± 0.41, 6.18 ± 1.03, 1.80 ± 0.71, 1.77 ± 0.55, 2.42 ± 0.71. Furthermore, gene transcription fold changes (hydrodynamic/control) increased linearly with TE corneal endothelium cells population morphology with r = 0.83 for TJP1 (ZO-1) and r = 0.58 for CTNND1 (p120-ctn). In fact, the more elongated the cells populations were, the greater hydrodynamic conditions increased the transcription of TJP1 (ZO-1) and CTNND1 (p120-ctn). These results suggest that hydrodynamic forces contribute to the maintenance of tight and adherens junctions of native corneal endothelial cells, as well as to the formation of tight and adherens junctions of corneal endothelial cells that are in the process of forming a functional endothelial barrier.

Immunohistochemical study of dissociation and association of adherens junctions in splenic sinus endothelial cells.

It is not yet clear whether cellular junctions between splenic sinus endothelial cells are open or closed. In order to clarify this, immunolocalization of thrombomodulin (TM), endothelial protein C receptor (EPCR), protease-activated receptor 1 (PAR1), sphingosine 1-phosphate receptor 1 (S1P1), β-catenin phosphorylated at Try142 (β-catenin Y142) and β-catenin phosphorylated at Try654 (β-catenin Y654), which are related proteins that regulate dissociation and association of the adherens junctions of endothelial cells, are examined in rats using laser microscopy and electron microscopy. TM, EPCR, PAR1 and S1P1 were colocalized in the entire circumference of the endothelial cells, as well as in the caveolar membranes and junctional membranes of adjacent endothelial cells. These molecules may protect the adherens junctions of the endothelial cells. On the other hand, β-catenin Y142 and β-catenin Y 654 colocalized with α-catenin and β-catenin, respectively and in addition, β-catenin Y142 and β-catenin Y 654 were localized in the vicinity of the adherens junctions of the endothelial cells from immunogold electron microcopy. The adherens junctions are considered to be partially dissociated at the site where β-catenin Y142 and β-catenin Y 654 are localized. Thus, the system that protects the adherens junctions and the system that dissociates them may concurrently coexist in the endothelial cells and dissociation and association of the adherens junctions may be constantly repeated at the cell boundary of the endothelial cells.

TM4SF19 aggravates LPS-induced attenuation of vascular endothelial cell adherens junctions by suppressing VE-cadherin expression

Atherosclerosis is a chronic vascular inflammatory disease that initially starts from an arterial intima lesion and endothelial barrier dysfunction. The purpose of this study was to investigate the role of TM4SF19, a recently identified member of the transmembrane 4L six superfamily, in vascular endothelial cell adherens junctions. We found TM4SF19 expression was significantly increased in atherosclerotic plaques and sera of patients with coronary heart disease (CHD) compared with healthy people by immunohistochemistry and ELISA. In vitro, human umbilical vein endothelial cells (HUVECs) were stimulated by lipopolysaccharides (LPS). TM4SF19 and VE-cadherin expression as well as cell adherens junctions were assessed. Additionally, LPS could upregulate TM4SF19 expression and downregulate VE-cadherin expression in HUVECs in a concentration dependent manner. Overexpression of TM4SF19 substantially aggravated LPS-induced reduction of VE-cadherin expression and attenuation of vascular endothelial cell adherens junctions. However, both the decreased VE-cadherin expression and weakened cell adherens junctions induced by LPS could be dramatically reversed when the expression of TM4SF19 was depressed. This study is the first to reveal the effect of TM4SF19 on endothelial cell adherens junctions. Meanwhile, our results also provide novel therapeutic strategies for atherosclerotic diseases.

MARCH Family E3 Ubiquitin Ligases Selectively Downregulate Cadherin Family Proteins

VE‐cadherin (VE‐cad) plays a central role in the adherens junctions (AJ) of vascular endothelial cells by regulating endothelial barrier function. Our group previously showed that a viral ubiquitin ligase K5, which is encoded in Human Herpesvirus 8 genome, causes endocytosis and degradation of VE‐cad. K5 is homologous to endogenous human membrane associated RING‐CH‐type ubiquitin ligase (MARCH) proteins. However, the contribution of endogenous MARCH family proteins to cadherin regulation is unclear. We began by performing RT‐PCR analysis to assess which MARCH family proteins are expressed in endothelial cells. Vascular endothelial cells were found to express low levels of MARCH1 and readily detectable levels of MARCH2, MARCH3, MARCH4 and MARCH8. To assess the effect of MARCH family protein expression on VE‐cad and N‐cad, GFP‐tagged MARCH family proteins were expressed in human umbilical endothelial cells (HUVECs) and the effect on VE‐cad and N‐cad was assessed. MARCH1 had no discernable impact on either VE‐cad or N‐cad. MARCH8 slightly downregulated VE‐cad but not N‐cad. In contrast, MARCH2, MARCH3 and MARCH4 transfection markedly downregulated VE‐cad in HUVECs. Interestingly, MARCH2 and MARCH3 did not downregulate N‐cad, but MARCH4 downregulated both VE‐cad and N‐cad. These results suggest that different MARCH proteins have different cadherin specificities. To determine the amino‐acid sequences within VE‐cad that dictate this specificity, we generated VE‐cad/N‐cad chimeras by swapping the transmembrane and juxta‐membrane domains (VE‐cad‐NcadTMDJMD, N‐cad‐VEcadTMDJMD). Interestingly, MARCH2 downregulated N‐cad‐VEcadTMDJMD but not VE‐cad‐NcadTMDJMD. These results indicate that the amino acid sequence within the transmembrane and juxta‐membrane domains is essential for the selectivity of MARCH2‐induced downregulation. These findings indicate that MARCH ubiquitin ligases exhibit selectivity for different members of the classical cadherin family. These findings suggest that MARCH family ligases may differentially regulate cadherins in vascular endothelial cells to modulate endothelial cell migration and barrier function during development and inflammation.

Excess centrosomes disrupt vascular lumenization and endothelial cell adherens junctions

Excess centrosomes disrupt vascular lumenization and endothelial cell adherens junctions

Adenosine:Adenosine Receptor (AR) Axis in Blood Brain Barrier (BBB) Permeability Regulation

Abstract Multiple sclerosis (MS) is a is a chronic, debilitating, neuroinflammatory disease that affects the central nervous system (CNS). MS affects about 2.5 million patients world-wide. MS patients experience a progressive loss in neurological function caused by immune-mediated axonal damage and demyelination in multiple areas of the brain and spinal cord. Despite years of research, the etiology of MS is still unknown. Studies using the dominant MS animal model, experimental autoimmune encephalomyelitis (EAE), demonstrate that myelin antigen-specific CD4 T cells can induce CNS inflammation, demyelination, and neurodegeneration, resulting in the loss of motor function (paralysis). Development of MS or EAE requires the entry of autoreactive immune cells into the CNS. Typically, the anatomical characteristics of the brain, such as the specialized endothelial cell tight and adherens junctions constitute the framework of the BBB, and limit the entry of lymphocytes into the CNS. We hypothesized that adenosine and/or adenosine receptors (ARs) expressed on BBB endothelial cells regulate the entry of immune cells and molecules into the brain. We showed that deletion of the adenosine-generating enzyme, CD73 (cd73−/−) expressed on BBB endothelial cells blocked immune cells entry into the brain. However, fulminant EAE could be induced in cd73(−/−) mice after the transfer of WT CD73(+)CD4(+) T cells, suggesting that CD73 must be expressed either on T cells or on BBB endothelial cells for disease induction. Next, we showed that blockade of the A2A AR block the entry of immune cells into the brain. Based on these findings we conclude that CD73 expression and adenosine receptor signaling are required for the efficient entry of lymphocytes into the CNS.

Empagliflozin Ameliorates Type 2 Diabetes-Induced Ultrastructural Remodeling of the Neurovascular Unit and Neuroglia in the Female db/db Mouse

Type 2 diabetes is associated with diabetic cognopathy. Anti-hyperglycemic sodium glucose transporter 2 (SGLT2) inhibitors have shown promise in reducing cognitive impairment in mice with type 2 diabetes mellitus. We recently described marked ultrastructural (US) remodeling of the neurovascular unit (NVU) in type 2 diabetic db/db female mice. Herein, we tested whether the SGLT-2 inhibitor, empagliflozin (EMPA), protects the NVU from abnormal remodeling in cortical gray and subcortical white matter. Ten-week-old female wild-type and db/db mice were divided into lean controls (CKC, n = 3), untreated db/db (DBC, n = 3), and EMPA-treated db/db (DBE, n = 3). Empagliflozin was added to mouse chow to deliver 10 mg kg−1 day−1 and fed for ten weeks, initiated at 10 weeks of age. Brains from 20-week-old mice were immediately immersion fixed for transmission electron microscopic study. Compared to CKC, DBC exhibited US abnormalities characterized by mural endothelial cell tight and adherens junction attenuation and/or loss, pericyte attenuation and/or loss, basement membrane thickening, glia astrocyte activation with detachment and retraction from mural cells, microglia cell activation with aberrant mitochondria, and oligodendrocyte–myelin splitting, disarray, and axonal collapse. We conclude that these abnormalities in the NVU were prevented in DBE. Empagliflozin may provide neuroprotection in the diabetic brain.

SENCR stabilizes vascular endothelial cell adherens junctions through interaction with CKAP4

SENCR is a human-specific, vascular cell-enriched long-noncoding RNA (lncRNA) that regulates vascular smooth muscle cell and endothelial cell (EC) phenotypes. The underlying mechanisms of action of SENCR in these and other cell types is unknown. Here, levels of SENCR RNA are shown to be elevated in several differentiated human EC lineages subjected to laminar shear stress. Increases in SENCR RNA are also observed in the laminar shear stress region of the adult aorta of humanized SENCR-expressing mice, but not in disturbed shear stress regions. SENCR loss-of-function studies disclose perturbations in EC membrane integrity resulting in increased EC permeability. Biotinylated RNA pull-down and mass spectrometry establish an abundant SENCR-binding protein, cytoskeletal-associated protein 4 (CKAP4); this ribonucleoprotein complex was further confirmed in an RNA immunoprecipitation experiment using an antibody to CKAP4. Structure-function studies demonstrate a noncanonical RNA-binding domain in CKAP4 that binds SENCR Upon SENCR knockdown, increasing levels of CKAP4 protein are detected in the EC surface fraction. Furthermore, an interaction between CKAP4 and CDH5 is enhanced in SENCR-depleted EC. This heightened association appears to destabilize the CDH5/CTNND1 complex and augment CDH5 internalization, resulting in impaired adherens junctions. These findings support SENCR as a flow-responsive lncRNA that promotes EC adherens junction integrity through physical association with CKAP4, thereby stabilizing cell membrane-bound CDH5.

Developmental SMAD6 loss leads to blood vessel hemorrhage and disrupted endothelial cell junctions

The BMP pathway regulates developmental processes including angiogenesis, yet its signaling outputs are complex and context-dependent. Recently, we showed that SMAD6, an intracellular BMP inhibitor expressed in endothelial cells, decreases vessel sprouting and branching both in vitro and in zebrafish. Genetic deletion of SMAD6 in mice results in poorly characterized cardiovascular defects and lethality. Here, we analyzed the effects of SMAD6 loss on vascular function during murine development. SMAD6 was expressed in a subset of blood vessels throughout development, primarily in arteries, while expression outside of the vasculature was largely confined to developing cardiac valves with no obvious embryonic phenotype. Mice deficient in SMAD6 died during late gestation and early stages of postnatal development, and this lethality was associated with vessel hemorrhage. Mice that survived past birth had increased branching and sprouting of developing postnatal retinal vessels and disorganized tight and adherens junctions. In vitro, knockdown of SMAD6 led to abnormal endothelial cell adherens junctions and increased VE-cadherin endocytosis, indicative of activated endothelium. Thus, SMAD6 is essential for proper blood vessel function during murine development, where it appears to stabilize endothelial junctions to prevent hemorrhage and aberrant angiogenesis.

Atorvastatin enhances endothelial adherens junctions through promoting VE-PTP gene transcription and reducing VE-cadherin-Y731 phosphorylation

Vascular endothelial protein tyrosine phosphatase (VE-PTP) is essential for endothelial cells (ECs) adherens junction and vascular homeostasis; however, the regulatory mechanism of VE-PTP transcription is unknown, and a drug able to promote VE-PTP expression in ECs has not yet been reported in the literature. In this study, we used human ECs as a model to explore small molecule compounds able to promote VE-PTP expression, and found that atorvastatin, a HMG-CoA reductase inhibitor widely used in the clinic to treat hypercholesterolemia-related cardiovascular diseases, strongly promoted VE-PTP transcription in ECs through activating the VE-PTP promoter and upregulating the expression of the transcription factor, specificity protein 1 (SP1). Additionally, atorvastatin markedly reduced VE-cadherin-Y731 phosphorylation induced by cigarette smoke extract and significantly enhanced stability of endothelial adherens junctions. Together, our findings reveal that atorvastatin up-regulates VE-PTP expression, increases VE-cadherin protein levels, and decreases VE-cadherin-Y731 phosphorylation to strengthen EC adherens junctions and maintain vascular cell monolayer integrity, offering a new mechanism of atorvastatin against CSE-induced disruption of vascular integrity and relevant cardio-cerebrovascular disease.

Endothelial BMP4 Regulates Leukocyte Diapedesis and Promotes Inflammation

Leukocyte recruitment is a fundamental event in the response of the innate immune system to injury. This process is promoted in part by the opening of endothelial cell adherens junctions that allows leukocyte extravasation through gaps between adjacent endothelial cells. VE-cadherin is a key component of endothelial cell adherens junctions and a negative regulator of leukocyte emigration. Accumulating evidence implicates bone morphogenetic protein (BMP) 4 as a critical regulator in vascular biology, but its role in leukocyte extravasation in vitro and in vivo has not been investigated so far. To assess the impact of BMP4 on leukocyte emigration in vivo, we used the thioglycollate-induced peritonitis model. C57BL/6 mice were intraperitoneally (i.p.) injected with recombinant BMP4 in addition to thioglycollate. Compared to solvent-treated controls, we observed higher accumulation of leukocytes in the peritoneal lavage of BMP4-treated mice indicating that BMP4 promotes leukocyte diapedesis into the inflamed peritoneal cavity. Endothelial cell-specific deletion of BMP4 in mice markedly diminished leukocyte diapedesis following thioglycollate administration suggesting that endothelial BMP4 is required for leukocyte recruitment. Consistent with these in vivo results, transwell migration assays with human umbilical vein endothelial cells (HUVECs) in vitro revealed that recombinant BMP4 enhanced leukocyte transmigration through the endothelial monolayer. Conversely, silencing of endothelial BMP4 by siRNA dampened leukocyte diapedesis in vitro. Mechanistic studies showed that loss of BMP4 improved endothelial junction stability by upregulation of VE-cadherin expression in vitro and in vivo. Vice versa, treatment of HUVECs with recombinant BMP4 decreased expression of VE-cadherin and impaired endothelial junction stability shown by Western blotting and immunocytochemistry. Finally, severe endothelial damage in HUVECs in response to serum of patients collected 24h after survived cardiac arrest was accompanied by increase in leukocyte migration in transwell assays and activation of the BMP pathway most probably by upregulation of endothelial BMP4 RNA and protein expression. Collectively, the present study provides novel evidence that endothelial BMP4 controls leukocyte recruitment through a VE-cadherin-dependent mechanism and that BMP4-induced inflammation might be involved in the pathogenesis of endothelial cell damage following successful resuscitation after cardiac arrest.

VE-Cadherin Disassembly and Cell Contractility in the Endothelium are Necessary for Barrier Disruption Induced by Tumor Cells

During metastasis, breakdown of the endothelial barrier is critical for tumor cell extravasation through blood vessel walls and is mediated by a combination of tumor secreted soluble factors and receptor-ligand interactions. However, a complete mechanism governing tumor cell transendothelial migration remains unclear. Here, we investigate the roles of tumor-associated signals in regulating endothelial cell contractility and adherens junction disassembly leading to endothelial barrier breakdown. We show that Src mediates VE-cadherin disassembly in response to metastatic melanoma cells. Through the use of pharmacological inhibitors of cytoskeletal contractility we find that endothelial cell contractility is responsive to interactions with metastatic cancer cells and that reducing endothelial cell contractility abrogates migration of melanoma cells across endothelial monolayers. Furthermore, we find that a combination of tumor secreted soluble factors and receptor-ligand interactions mediate activation of Src within endothelial cells that is necessary for phosphorylation of VE-cadherin and for breakdown of the endothelial barrier. Together, these results provide insight into how tumor cell signals act in concert to modulate cytoskeletal contractility and adherens junctions disassembly during extravasation and may aid in identification of therapeutic targets to block metastasis.