YES kinase controls endothelial junctional plasticity and barrier integrity by regulating VE-cadherin phosphorylation and endocytosis

Vascular Endothelial (VE)-cadherin in endothelial adherens junctions is an essential component of the vascular barrier, critical for tissue homeostasis and implicated in progression of diseases such as cancer and eye diseases. Inhibitors of SRC cytoplasmic tyrosine kinase have been applied to suppress tyrosine phosphorylation of VE-cadherin and thereby to prevent excessive leakage, edema and high interstitial pressure. We show that the SRC-related YES tyrosine kinase rather than SRC, is localized at endothelial cell (EC) junctions. EC-specific YES deletion suppresses VE-cadherin phosphorylation, and arrests VE-cadherin at EC junctions. This is accompanied by loss of EC collective migration, and exaggerated agonist-induced macromolecular leakage, while extravasation of monocytes is suppressed. Overexpression of Yes causes ectopic VE-cadherin phosphorylation while vascular leakage is unaffected. In contrast, in EC-specific Src-deficient mice, VE-cadherin internalization is maintained and leakage is suppressed. In conclusion, YES-mediated VE-cadherin phosphorylation regulates its constitutive turnover, required for endothelial junction plasticity and vascular integrity.

Sphingosine Kinase 1 Plays an Important Role in Atorvastatin-Mediated Anti-Inflammatory Effect against Acute Lung Injury

Atorvastatin is a 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) inhibitor and inhibits cholesterol synthesis. Recently, atorvastatin also showed anti-inflammatory effect in acute lung injury, ameliorating pulmonary gas-blood exchanging function. Sphingosine kinase 1 plays a central role in endothelial (EC) cytoskeleton rearrangement and EC barrier integrity regulation. In this study, the role of sphingosine kinase 1 in atorvastatin anti-inflammatory effect against acute lung injury was investigated. Both wild-type (WT) and SphK1-/- mice were challenged with high tidal volume ventilation (40 ml/kg body weight, 65 breathing/min, 4 hours). The acute lung injury was evaluated and the mechanisms were explored. In WT mice, atorvastatin treatment significantly decreased acute lung injury responding to high tidal volume ventilation (HT), including protein, cellular infiltration, and cytokine releasing; comparing to WT mice, SphK1-/- mice showed significantly worsen pulmonary injuries on HT model. Moreover, the atorvastatin-mediated anti-inflammatory effect was diminished in SphK1-/- mice. To further confirm the role of SphK1 in VILI, we then compared the inflammatory response of endothelial cells that were isolated from WT and SphK1-/- mice to cyclic stretching. Similarly, atorvastatin significantly decreased cytokine generation from WT EC responding to cyclic stretching. Atorvastatin also significantly preserved endothelial junction integrity in WT EC against thrombin challenge. However, the inhibitory effect of atorvastatin on cytokine generation induced by cyclic stretching was abolished on SphK1-/- mice EC. The endothelial junction integrity effects of atorvastatin also diminished on SphK1-/- mouse EC. Signal analysis indicated that atorvastatin inhibited JNK activation induced by cyclic stretch. SphK1 knockout also blocked atorvastatin-mediated VE-cadherin junction enhancement. In summary, by inhibition of MAPK activity and maintenance of EC junction homeostasis, SphK1 plays a critical role in atorvastatin-mediated anti-inflammatory effects in both cellular and in vivo model. This study also offers an insight into mechanical stress-mediated acute lung injury and potential therapy in the future.

Hidrox ® and Chronic Cystitis: Biochemical Evaluation of Inflammation, Oxidative Stress, and Pain

Interstitial cystitis/painful bladder syndrome (IC/PBS) is a chronic bladder condition characterized by frequent urination, inflammation, oxidative stress, and pain. The aim of the study was to evaluate the anti-inflammatory and antioxidant effects of an oral administration of Hidrox® (10 mg/kg) in the bladder and spinal cord in a rodent model of IC/BPS. The chronic animal model of cystitis was induced by repeated intraperitoneal injections of cyclophosphamide (CYP) for five consecutive days. Treatment with Hidrox ® began on the third day of the CYP injection and continued until the 10th day. CYP administration caused macroscopic and histological bladder changes, inflammatory infiltrates, increased mast cell numbers, oxidative stress, decreased expression of the tight endothelial junction (e.g., zonula occludens-1 (ZO-1) and occludin), and bladder pain. Treatment with Hidrox® was able to improve CYP-induced inflammation and oxidative stress via the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1) pathway. It was also able to reduce bladder pain which was aggravated by the activation of neuroinflammation in the central nervous system. In particular, Hidrox® reduced the brain-derived neurotrophic factor (BDNF), as well as the activation of astrocytes and microglia, consequently reducing mechanical allodynia. These results indicate that nutritional consumption of Hidrox® can be considered as a new therapeutic approach for human cystitis, increasing the conceivable potential of a significant improvement in the quality of life associated with a lowering of symptom intensity in patients with IC/BPS.

Hepatocyte Growth Factor Protects Endothelial Barrier Against Oxidative Stress and Mitochondria-dependent Apoptosis

Background: Pulmonary microvascular endothelial cells(PMVECs) were incomplex and endothelial barrier was destroyed in the pathogenesis progress of acute lung injury(ALI)/acute respiratory distress syndrome(ARDS). Previous studies have demonstrated that hepatocyte growth factor (HGF) could decrease endothelial apoptosis. Nevertheless, the mechanism by which HGF-suppressed oxidative stress contributes to endothelial mitochondria-dependent apoptosis is poorly understood. Methods: In our current study, we introduced lipopolysaccharide(LPS)-induced PMEVCs with HGF treatment. To investigate the effects of mTOR/STAT-3 pathway in endothelial oxidative stress and mitochondria-dependent apoptosis, mammalian TOR (mTOR) inhibitor rapamycin and signal transducer and activator of transcription 3 (STAT-3) inhibitor S3I-201 were respectively used to inhibit mTOR/STAT-3 signaling. Moreover, lentivirus vector-mediated HGF, mTORC1(raptor) and mTORC2(rictor) gene knockdown modification were introduced to evaluate mTORC1 and mTORC2 pathway. Calcium measurement, ROS production, mitochondrial membrane potential and protein complex I expression, cell proliferation, apoptosis and endothelial junction protein were detected to evaluate HGF effects.Results: Our study demonstrated that HGF protected endothelium via the suppression of ROS production and intracellular calcium uptake, which leading to increased mitochondrial membrane potential (JC-1 and mitochondria tracker green detection)and specific proteins(complex I), decreased endothelial apoptosis specific protein(Caspase-3), raised anti-apoptosis mRNA level(Bcl-2 and Bcl-xL), and increased endothelial junction proteins (VE-cadherin and occludin). Reversely, mTOR inhibitor rapamycin and STAT-3 inhibitor S3I-201 could raise oxidative stress and mitochondria-dependent apoptosis even with HGF treatment in LPS-induced endothelial cells. Similarly, mTORC1 as well as mTORC2, have the same protective effects in mitochondria damage and apoptosis. Conclusion: In all, these reveal that mTOR/STAT-3 signaling mediate the HGF suppression effects to oxidative level, mitochondria-dependent apoptosis and endothelial junction protein in LPS-stimulated PMVECs, which contributing to the endothelial survival and barrier integrity.

Neutrophil transendothelial migration hotspots – mechanisms and implications

ABSTRACTDuring inflammation, leukocytes circulating in the blood stream exit the vasculature in a process called leukocyte transendothelial migration (TEM). The current paradigm of this process comprises several well-established steps, including rolling, adhesion, crawling, diapedesis and sub-endothelial crawling. Nowadays, the role of the endothelium in transmigration is increasingly appreciated. It has been established that leukocyte exit sites on the endothelium and in the pericyte layer are in fact not random but instead may be specifically recognized by migrating leukocytes. Here, we review the concept of transmigration hotspots, specific sites in the endothelial and pericyte layer where most transmigration events take place. Chemokine cues, adhesion molecules and membrane protrusions as well as physical factors, such as endothelial junction stability, substrate stiffness, the presence of pericytes and basement membrane composition, may all contribute to local hotspot formation to facilitate leukocytes exiting the vasculature. In this Review, we discuss the biological relevance of such hotspots and put forward multiple mechanisms and factors that determine a functional TEM hotspot.

Vascular Endothelial Integrity Affects the Severity of Enterovirus-Mediated Cardiomyopathy

Coxsackievirus and adenovirus receptor (CAR) is present in epithelial and vascular endothelial cell junctions. We have previously shown a hemorrhagic phenotype in germ-line CAR knock-out mouse embryos; we have also found that CAR interacts with ZO-1 and β-catenin. However, the role of CAR in vascular endothelial junction permeability has not been proven. To understand the roles of CAR in the vascular endothelial junctions, we generated endothelium-specific CAR knockout (CAR-eKO) mice. In the absence of CAR, the endothelial cell layer showed an increase in transmembrane electrical resistance (TER, Ω) and coxsackievirus permeability. Evans blue dye and 70 kDa dextran-FITC were delivered by tail vein injection. We observed increased vascular permeability in the hearts of adult CAR-eKO mice compare with wild-type (WT) mice. There was a marked increase in monocyte and macrophage penetration into the peritoneal cavity caused by thioglycolate-induced peritonitis. We found that CAR ablation in endothelial cells was not significantly increased coxsackievirus B3 (CVB3) induced myocarditis in murine model. However, tissue virus titers were significantly higher in CAR-eKO mice compared with WT. Moreover, CVB3 was detected in the brain of CAR-eKO mice. Endothelial CAR deletion affects the expression of major endothelial junction proteins, such as cadherin and platelet endothelial cell adhesion molecule-1 (PECAM-1) in the cultured endothelial cells as well as liver vessel. We suggest that CAR expression is required for normal vascular permeability and endothelial tight junction homeostasis. Furthermore, CVB3 organ penetration and myocarditis severities were dependent on the endothelial CAR level.

Modeling the three-way feedback between cellular contractility, actin polymerization, and adhesion turnover resolves the contradictory effects of RhoA and Rac1 on endothelial junction dynamics

The formation and recovery of gaps in the vascular endothelium governs a wide range of physiological and pathological phenomena, from angiogenesis to atherosclerosis and tumor cell extravasation. However, the interplay between the mechanical and signaling processes that drive dynamic behavior in vascular endothelial cells is not well understood. In this study, we propose a chemo-mechanical model to investigate the maintenance of endothelial junctions as dependent on the crosstalk between actomyosin contractility, VE-cadherin bond turnover, and actin polymerization, which mediate the forces exerted on the cell-cell interface. Our theoretical model reveals that active cell tension can stabilize cadherin bonds within an adhesion, but excessive RhoA signaling can drive bond dissociation and junction failure. While Rac1-mediated actin polymerization aids gap closure, high levels of Rac1 may also facilitate junction weakening. Combining the modeling framework with novel experiments, we identify how dynamic rupture and heal cycles emerge and, further, describe why gaps tend to localize at multi-cell contacts. Beyond, our analysis also indicates that a critical balance between RhoA and Rac1 expression is required to maintain junction stability and limit endothelial dysfunction. The model predicts how pharmacological modulation of actin polymerization and cell contractility impacts junction stability, with predictions subsequently validated experimentally. Our proposed framework can help guide the development of therapeutics that target the Rho family of GTPases and downstream active mechanical processes.

Endothelial Regulation by Exogenous Annexin A1 in Inflammatory Response and BBB Integrity Following Traumatic Brain Injury

Background and TargetFollowing brain trauma, blood–brain barrier (BBB) disruption and inflammatory response are critical pathological steps contributing to secondary injury, leading to high mortality and morbidity. Both pathologies are closely associated with endothelial remodeling. In the present study, we concentrated on annexin A1 (ANXA1) as a novel regulator of endothelial function after traumatic brain injury.MethodsAfter establishing controlled cortical impact (CCI) model in male mice, human recombinant ANXA1 (rANXA1) was administered intravenously, followed by assessments of BBB integrity, brain edema, inflammatory response, and neurological deficits.ResultAnimals treated with rANXA1 (1 μg/kg) at 1 h after CCI exhibited optimal BBB protection including alleviated BBB disruption and brain edema, as well as endothelial junction proteins loss. The infiltrated neutrophils and inflammatory cytokines were suppressed by rANXA1, consistent with decreased adhesive and transmigrating molecules from isolated microvessels. Moreover, rANXA1 attenuated the neurological deficits induced by CCI. We further found that the Ras homolog gene family member A (RhoA) inhibition has similar effect as rANXA1 in ameliorating brain injuries after CCI, whereas rANXA1 suppressed CCI-induced RhoA activation.ConclusionOur findings suggest that the endothelial remodeling by exogenous rANXA1 corrects BBB disruption and inflammatory response through RhoA inhibition, hence improving functional outcomes in CCI mice.