Endothelial Cell Disruption Research Articles

Increased claudin‐3, ‐4 and ‐18 levels in bronchoalveolar lavage fluid reflect severity of acute lung injury

Acute lung injury (ALI) is characterized by disruption of lung epithelial and endothelial cells, leading to increased membrane permeability and loss of barrier function. Claudins are key components of tight junctions (TJ) that regulate paracellular permeability, and play an important role in alveolar epithelial barrier function and fluid clearance. However, whether claudin-3, -4, -18 or -5 expression changes in Pseudomonas aeruginosa (PA)-induced ALI and the clinical significance of such change is unknown. Rats underwent intratracheal instillation of PA, and samples were collected prior to and 3, 9 and 24 h after instillation. Lung injury was evaluated by bronchoalveolar lavage fluid (BALF) total protein, arterial blood gas analysis, lung injury score, and expression of surfactant protein and von Willebrand factor. Claudin expression in lung was measured with quantitative real-time polymerase chain reaction and western blotting, and in BALF by enzyme-linked immunosorbent assay. The relationship between claudins in BALF and lung injury grade were analysed with Spearman's rank correlation. Alveolar epithelium, endothelium and TJ ultrastructure were observed with electron microscopy. Claudin-4, -18 and -5 mRNA levels increased significantly 24 h after PA instillation in the most severe lung injury cases, whereas there was no significant change in protein levels. Claudin-3, -4 and -18 levels in BALF increased most 24 h after PA instillation; this paralleled alveolar epithelial disruption and lung injury severity. Claudin-3, -4 and -18 released into the alveolar compartment is highly associated with barrier function loss caused by alveolar epithelial injury.

P67: Role of oxidative stress-induced caveolin-1 S-nitrosylation, ubiquitination, and degradation in endothelial cell dedifferentiation and idiopathic pulmonary arterial hypertension

Cav1 −/− mice develop pulmonary arterial hypertension (PAH) which is dependent on eNOS since inhibition or deletion of eNOS abrogates the PAH phenotype. Here, we tested the hypothesis that inflammatory signaling/oxidative stress induces Cav-1 degradation, and that in presence of reduced Cav-1 expression, eNOS hyperactivation promotes endothelial cell dedifferentiation, disorganized postnatal angiogenesis, and vaso-occlusive disease in the lung. In freshly-isolated IPAH patient-derived lung endothelial cells, we observed reduced Cav-1 expression and eNOS hyperphosphorylation. In cultured control human lung endothelial cells, TNF alpaha-induced NO production and S -nitrosylation (SNO) of Cav-1 cysteine 156 was associated with increased Src activity and destabilization of Cav-1 oligomeric chains that could be blocked by the eNOS inhibitor l -NAME or Src inhibitor PP2. Furthermore, Cav-1 SNO was associated with reduced Cav-1 expression following prolonged (72 h) stimulation with TNF alpha or the NO donor DetaNONOate. Moreover, mass spectrometry revealed K86 in the caveolin scaffold domain as a site of Cav-1 ubiquitination (Ub). Thus, these data suggest that Cav-1 SNO at C156 induces Src-dependent Cav-1 phosphorylation at Y14, destabilization of Cav1 oligomers, and Ub of K86 leading to Cav-1 degradation. Furthermore, Cultured Cav1 −/− ECs were observed to be hyperproliferative and dysfunctional in their ability to generate lumenized vessels in Matrigel plugs in vitro and in vivo. The defect in angiogenesis observed in Cav1 −/− ECs was shown to be due to hyperactive eNOS-mediated disruption of endothelial cell–cell junctions and cell contact-dependent Notch signaling. eNOS-dependent peroxynitrite production and resultant Akt and Src hyperactivation in Cav1 −/− ECs not only prevented nuclear Notch signaling, it resulted in increased nuclear beta-catenin signaling that was associated with an “all tip-cell” phenotype. Rescue of Cav-1 null cells with Cav-1 cDNA and in Cav1 −/− × eNOS −/− double knockout ECs, Notch activation and angiogenesis were restored. Therefore, oxidative stress-induced Cav-1 degradation and eNOS hyperactivation-mediated endothelial cell dedifferentiation may play an important role in vaso-occlusive pulmonary vascular disease. Disclosure Nothing to disclose.

Hydrogen sulfide dilates rat mesenteric arteries by activating endothelial large-conductance Ca2+-activated K+ channels and smooth muscle Ca2+ sparks

We have previously shown that hydrogen sulfide (H₂S) reduces myogenic tone and causes relaxation of phenylephrine (PE)-constricted mesenteric arteries. This effect of H₂S to cause vasodilation and vascular smooth muscle cell (VSMC) hyperpolarization was mediated by large-conductance Ca(2+)-activated potassium channels (BKCa). Ca(2+) sparks are ryanodine receptor (RyR)-mediated Ca(2+)-release events that activate BKCa channels in VSMCs to cause membrane hyperpolarization and vasodilation. We hypothesized that H₂S activates Ca(2+) sparks in small mesenteric arteries. Ca(2+) sparks were measured using confocal microscopy in rat mesenteric arteries loaded with the Ca(2+) indicator fluo-4. VSMC membrane potential (Em) was measured in isolated arteries using sharp microelectrodes. In PE-constricted arteries, the H₂S donor NaHS caused vasodilation that was inhibited by ryanodine (RyR blocker), abluminal or luminal iberiotoxin (IbTx, BKCa blocker), endothelial cell (EC) disruption, and sulfaphenazole [cytochrome P-450 2C (Cyp2C) inhibitor]. The H₂S donor NaHS (10 μmol/l) increased Ca(2+) sparks but only in the presence of intact EC and this was blocked by sulfaphenazole or luminal IbTx. Inhibiting cystathionine γ-lyase (CSE)-derived H2S with β-cyano-l-alanine (BCA) also reduced VSMC Ca(2+) spark frequency in mesenteric arteries, as did EC disruption. However, excess CSE substrate homocysteine did not affect spark activity. NaHS hyperpolarized VSMC Em in PE-depolarized mesenteric arteries with intact EC and also hyperpolarized EC Em in arteries cut open to expose the lumen. This hyperpolarization was prevented by ryanodine, sulfaphenazole, and abluminal or luminal IbTx. BCA reduced IbTx-sensitive K(+) currents in freshly dispersed mesenteric ECs. These results suggest that H₂S increases Ca(2+) spark activity in mesenteric artery VSMC through activation of endothelial BKCa channels and Cyp2C, a novel vasodilatory pathway for this emerging signaling molecule.

Lymphocyte infiltration and activation in iris-ciliary body and anterior chamber of mice in corneal allograft rejection.

To investigate the infiltration and activation of lymphocyte in iris-ciliary body and anterior chamber after allogenic penetrating keratoplasty (PK), for further revealing the role of iris-ciliary body in corneal allograft immune rejection. In the mice models of PK, BALB/C mice received orthotopic isografts (n =35) or C57BL/6 donor allografts (n =25). Grafts were examined daily for 3 weeks by slit-lamp microscopy and scored for opacity. The infiltration of CD4(+) T lymphocyte in iris-ciliary body and anterior chamber was examined by immunohistology and the mRNA of CD80 and CD86 in both cornea graft and iris-ciliary body by RT-PCR was analyzed in allograft recipient at days 3, 6, 10 and the day when graft rejection occurred. Isograft recipients were examined as control at the corresponding time points. Transmission electron microscope was used to study the ultrastructure, especially cell infiltration, of iris-cilary body and corneal graft at day 3, 7 and the day when rejection occurred after allogenic PK. Rejection was observed in all the allograft recipients followed more than 10 days, at a median time of 15 days (range 12-18 days), but not in any of isografts. CD4(+) T cells were first detected at day 6 after transplantation in limbus and Ciliary body, and then in the stroma of recipient, iris, anterior chamber and corneal allograft with an increased number until graft rejection occurred. CD80 and CD86 mRNA were detected under RT-PCR examination in both graft and iris-ciliary body of allograft recipient, but not in any of isograft recipient. Three days after operation, lymphocytes and monocytes macrophages were visible in iris blood vessels and the anterior chamber, and vascular endothelial cell proliferation and activation were significant under transmission electron microscopy examination. At day 7, corneal endothelial cells became thinner. Lymphocytes and mononuclear macrophages were found with great number in the anterior chamber and adhered to the corneal endothelium. Blood vessels in iris increased and were filled with lymphocytes. And lymphocytes were detected to migrate through endothelial cell gap out of vessels. When allograft rejection occurred, macrophages attached to endothelial cells with large number of lymphocytes and macrophages infiltrating in iris. Lymphocyte infiltration and activation occurred in iris-ciliary body after allogenic PK, and the lymphocytes could migrate from iris blood vessel to the anterior chamber, which might play an important role in corneal allograft immune rejection.

Granzyme B cleavage of fibronectin disrupts endothelial cell adhesion, migration and capillary tube formation

Dysregulated angiogenesis contributes to the pathogenesis of chronic inflammatory diseases. Modulation of the extracellular matrix by immune-derived proteases can alter endothelial cell–matrix interactions as well as endothelial cell sprouting, migration and capillary formation. Granzyme B is a serine protease that is expressed by a variety of immune cells, and accumulates in the extracellular milieu in many chronic inflammatory disorders that are associated with dysregulated angiogenesis. Although granzyme B is known to cleave fibronectin, an essential glycoprotein in vascular morphogenesis, the role of granzyme B in modulating angiogenesis is unknown. In the present study, granzyme B cleaved both plasma fibronectin and cell-derived fibronectin, resulting in the release of multiple fibronectin fragments. Granzyme B cleavage of fibronectin resulted in a dose-dependent reduction in endothelial cell adhesion to fibronectin as well as reduced endothelial cell migration and tubular formation. These events were prevented when granzyme B activity was inhibited by a small molecule inhibitor. In summary, granzyme B-mediated cleavage of fibronectin contributes to attenuated angiogenesis through the disruption of endothelial cell — fibronectin interaction resulting in impaired endothelial cell migration and tubular formation.

C0363 Discoid platelet aggregations visualized by in vivo molecular imaging, and contribution of inflammatory cytokines

Background: The mechanism by which thrombotic vessel occlusion occurs independently of plaque development or endothelial cell (EC) disruption remains unclear, largely because of an inability to visualize thrombus formation, especially at the single-platelet level in real time. Methods: Therefore, we developed in vivo imaging technique based on singleand multi-photon microscopy, and we assessed dynamic cellular interplay in thrombosis models. We visualized that rapidly developing thrombi composed of discoid platelets without EC disruptionwas triggered by ROS photochemically inducedbymoderate power laser irradiation. Results: Using this technique, we elucidated that Lnk (adapter protein) regulates integrin signaling leading to stabilization of developing thrombus in vivo (2010 JCI). We established the efficient culture system of human iPS-derived platelets, and we confirmed the functional role. These artificial platelets can circulate, and contribute to their thrombus formation in vivo, indicating the clinical usefulness considering the cell therapy for future (2010 JEM). In addition, we elucidated the contribution of inflammatory cytokines, ROS, and integrin signaling to our thrombosis models (Nishimura et al, 2011 Blood). The inflammatory cytokines TNF-alpha and IL-1 could be key components of the EC response, acting through regulation of von Willebrand factor mobilization to the cell surface. Thrombus formation was then initiated by the binding of platelet GPIbalpha to endothelial vonWillebrand Factor in our model, and this effect was inhibited by the ROS scavenger N-acetylcysteine. Actin linker talindependent activation of alphaIIb-beta3 integrin in platelets was required for late phase thrombus stability. Comment: In sum, using our imaging system can be a powerful tool to analyze thrombus formation. We clarified the mechanism of discoid platelet aggregations on undisputed endothelium. The initial platelet aggregation subsequently leads to irreversible integrinand actindependent thrombus development. Inflammatory cytokine signaling in ECs also played pivotal role.

Abstract 637: Hydrogen Sulfide Acts on Endothelial Cells to Elicit Dilation.

Hydrogen sulfide (H 2 S), produced by the enzyme cystathionine-γ lyase (CSE), dilates arteries by hyperpolarizing and relaxing vascular smooth muscle cells (VSMC) and CSE knock-out causes hypertension and endothelial dysfunction showing the importance of this system. However, it is not clear if H 2 S-induced VSMC depolarization and relaxation is mediated by direct effects on VSMC or indirectly through actions on endothelial cells (EC). We reported previously that disrupting EC prevents H 2 S-induced vasodilation suggesting H 2 S might act directly on EC. Because inhibiting large-conductance Ca 2+ -activated K + (BK Ca ) channels also inhibits H 2 S-induced dilation, we hypothesized that H 2 S activates EC BK Ca channels to hyperpolarize EC and increase EC Ca 2+ which stimulates release of a secondary hyperpolarizing factor. Small mesenteric arteries from male Sprague-Dawley rats were used for all experiments. We found that EC disruption prevented H 2 S-induced VSMC membrane potential ( E m ) hyperpolarization. Blocking EC BK Ca channels with luminal application of the BK Ca inhibitor, iberiotoxin (IbTx, 100 nM), also prevented NaHS-induced dilation and VSMC hyperpolarization but did not affect resting VSMC E m showing EC specific actions. Sharp electrode recordings in arteries cut open to expose EC demonstrated H 2 S-induced hyperpolarization of EC while Ca 2+ imaging studies in fluor-4 loaded EC showed that H 2 S increases EC Ca 2+ event frequency. Thus H 2 S can act directly on EC. Inhibiting the EC enzyme cytochrome P 450 2C (Cyp2C) with sulfaphenazole also prevented VSMC depolarization and vasodilation. Finally, inhibiting TRPV4 channels to block the target of the Cyp2C product 11,12-EET inhibited NaHS-induced dilation. Combined with our previous report that CSE inhibition decreases BK Ca currents in EC, these results suggest that H 2 S stimulates EC BK Ca channels and activates Cyp2C upstream of VSMC hyperpolarization and vasodilation.

OS076. The effect of lectin-like oxidized low density lipoprotein receptor monoclonal antibody (LOX-1 MAb) on the expression of eNOS in preeclamptic HUVECs model

Preeclampsia is a major cause of fetal and maternal morbidity and mortality and it still occurs in 7-10% of all pregnancies worldwide. The pathophysiology remains unclear and so it was named "a disease of theories". Clinical and biochemical evidences assumed that disruption of normal endothelial cells may be the primary cause in the pathogenesis of preeclampsia. Factors that cause endothelial dysfunction are not certainly known, but the evidence suggests that factors related to the placenta. In a previous study conducted, HUVECs were found to significantly increase the uptaking of Ox-LDL by endothelial cells in response to the exposure to preeclampsia plasma when compared with non-pregnant women plasma and plasma of pregnant women. Ox-LDL binds to LOX-1 in vascular cells, including endothelial cells and smooth muscle cells, and is thought to cause endothelial dysfunction in patients with preeclampsia. One sign of endothelial dysfunction is the decline in biological activity of endothelial derived NO. NO biological half-time in vivo is less than 1 second. Nitric oxide is produced by the five-electron oxidation of L-arginine which is catalyzed by the enzyme eNOS. This research will observes the expression of eNOS in HUVECs PE model which were exposed to 5, 10, 20μg/ml LOX-1 MAb (lectin-like oxidized LDL receptor monoclonal antibody). To know the effect of LOX-1 MAb on the expression of eNOS in Preeclamptic HUVECs Model. The study used HUVECs PE model which is HUVECs culture that was exposed with pre-eclamptic serum and it shown endothelial dysfunction. HUVECs culture was taken from the endothel of umbilical cord of newborn. HUVECs culture were divided into four groups: (1) HUVECs + Preeclamptic serum (HUVECs PE model), (2) HUVECs PE Model + LOX-1 MAb 5μg/ml, (3) HUVECs PE Model + LOX-1 MAb 10;μg/ml, 4) HUVECs PE Model + LOX-1 MAb 20μg/ml. We performed immunocytochemistry study using primary antibody (anti eNOS) while eNOS expression was observed in each group. The data were analyzed using ANOVA, Tukey's test, Correlation and Regression test. eNOS expression in group 1 was significantly different with group 2, 3 and 4 (p<0.05). Group 2 was significantly different with group 1, 3 and 4 (p<0.05). In group 4 and 5 there were no significant difference each other, but both were significantly different with group 1 and 2 (p<0.05). About 72% of the expression of eNOS in HUVECs PE model were influenced by exposure LOX-1 MAb and the other 28% were influenced by other factors. In the observation of HUVECs PE model, we found that the higher dosage of LOX-1 MAb given on the model, the eNOS expression will increase proportionally. Exposure of LOX-1 MAb will significantly increase eNOS expression on HUVECs PE model. We concluded that the higher dosage of LOX-1 MAb given on the HUVECs PE model, the eNOS expression will increase proportionally.

Abstract 41: Discoid Platelet Aggregations Visualized by in Vivo Molecular Imaging Without Endothelium Disruption and Contribution of Inflammatory Cytokine, Ros and Integrin Signaling

inflammation, it is vital to examine the multi-cellular kinetics in living animals. Therefore, we developed in vivo imaging technique based on single- and multi-photon microscopy, and we assessed dynamic cellular interplay in diseased conditions. The mechanism by which thrombotic vessel occlusion occurs independently of plaque development or endothelial cell (EC) disruption remains unclear, largely because of an inability to visualize thrombus formation, especially at the single-platelet level in real time. Here we demonstrate that rapidly developing thrombi composed of discoid platelets can be induced in the mesenteric vessls of living mice, enabling characterization of the kinetics of thrombosis initiation and the multicellular interrelationships during thrombus development. Platelet aggregation without EC disruption was triggered by reactive oxygen species (ROS) photochemically induced by moderate power laser irradiation. Using this technique, we elucidated that Lnk (adapter protein) regulates integrin signaling leading to stabilization of developing thrombus in vivo (Nishimura et at, 2010 JCI). In addition, we established the efficient culture system of human iPS-derived platelets, and we confirmed the functional role. These artificial platelets can circulate, and contribute to their thrombus formation in vivo, indicating the clinical usefulness considering the cell therapy for future (Takayama &amp; Nishimmura et al, 2010 JEM). In addition, we elucidated the contribution of inflammatory cytokines, ROS, and integrin signaling to our thrombosis models (Nishimura et al, 2011 Blood). The inflammatory cytokines TNF-alpha and IL-1 could be key components of the EC response, acting through regulation of von Willebrand factor mobilization to the cell surface. Thrombus formation was then initiated by the binding of platelet GPIb-alpha to endothelial von Willebrand Factor in our model, and this effect was inhibited by the ROS scavenger N-acetylcysteine. Actin linker talin-dependent activation of alphaIIb-beta3 integrin in platelets was required for late phase thrombus stability. Our imaging can be applied to many research fields including metabolic diseases. Our imaging revealed close spatial and temporal interrelationships between angiogenesis and adipogenesis in obese adipose (Nishimura et al, 2007 Diabetes). In addition, increased inflammatory cell interactions were visualized (Nishimura et al, 2008 JCI). We also found that large numbers of CD8+ effector T cells infiltrated into obese adipose, and these cells were essential for the initiation and development of adipose inflammation (Nishimura et al, 2009 Nat Med). In sum, using our imaging system can be a powerful tools to analyze the inflammatory conditions including thrombosis. We clarified the mechanism by which thrombi are rapidly formed by discoid platelets on undisputed endothelium. The initial platelet aggregation subsequently lead to irreversible integrin- and actin-dependent thrombus development. Inflammatory cytokine signaling in ECs played pivotal role in discoid platelet aggregations in vivo.

In vivo imaging visualizes discoid platelet aggregations without endothelium disruption and implicates contribution of inflammatory cytokine and integrin signaling

AbstractThe mechanism by which thrombotic vessel occlusion occurs independently of plaque development or endothelial cell (EC) disruption remains unclear, largely because of an inability to visualize the formation of thrombus, especially at the single-platelet level in real time. Here we demonstrate that rapidly developing thrombi composed of discoid platelets can be induced in the mesenteric capillaries, arterioles, and large-sized arteries of living mice, enabling characterization of the kinetics of thrombosis initiation and the multicellular interrelationships during thrombus development. Platelet aggregation without EC disruption was triggered by reactive oxygen species (ROS) photochemically induced by moderate power laser irradiation. The inflammatory cytokines TNF-α and IL-1 could be key components of the EC response, acting through regulation of VWF mobilization to the cell surface. Thrombus formation was then initiated by the binding of platelet GPIbα to endothelial VWF in our model, and this effect was inhibited by the ROS scavenger N-acetylcysteine. Actin linker talin-dependent activation of alphaIIb-beta3 integrin or Rac1 in platelets was required for late-phase thrombus stability. Our novel imaging technology illustrates the molecular mechanism underlying inflammation-based thrombus formation by discoid platelets on undisrupted ECs and suggests control of ROS could be a useful therapeutic target for the prevention of thrombotic diseases.

Angiopoietin2 and Tie2: Tied to Lymphangiogenesis and Lung Metastasis. New Perspectives in Antimetastatic Antiangiogenic Therapy

Angiopoietin2 and Tie2: Tied to Lymphangiogenesis and Lung Metastasis. New Perspectives in Antimetastatic Antiangiogenic Therapy

PIGF Knockout Delays Brain Vessel Growth and Maturation upon Systemic Hypoxic Challenge

In this study, we have investigated the potential role of placental growth factor (PlGF) in hypoxia-induced brain angiogenesis. To this end, PlGF wild-type (PlGF(+/+)) and PlGF knockout (PlGF(-/-)) mice were exposed to whole body hypoxia (10% oxygen) for 7, 14, and 21 days. PlGF(+/+) animals exhibited a significant ~40% increase in angiogenesis after 7 days of hypoxia compared with controls, while in PlGF(-/-) this effect only occurred after 14 days of hypoxia. No differences in pericyte/smooth muscle cell (SMC) coverage between the two genotypes were observed. After 14 days of hypoxia, PlGF(-/-) microvessels had a significant increase in fibrinogen accumulation and extravasation compared with those of PlGF(+/+), which correlated with endothelial cell disruption of the tight junction protein claudin-5. These vessels displayed large lumens, were surrounded by reactive astrocytes, lacked both pericyte/SMC coverage and endothelial vascular endothelial growth factor expression, and regressed after 21 days of hypoxia. Vascular endothelial growth factor expression levels were found to be significantly lower in the frontal cortex of PlGF(-/-) compared with those in PlGF(+/+) animals during the first 5 days of hypoxia, which in combination with the lack of PlGF may have contributed to the delayed angiogenic response and the prothrombotic phenotype observed in the PlGF(-/-)animals.

Successful sirolimus rescue in tacrolimus‐induced thrombotic microangiopathy after living‐related liver transplantation

TMA is a rare complication of tacrolimus. Disruption of endothelial cells, platelet aggregation, and intravascular mechanical fragmentation of red cells are core mechanisms of injury; however, exact pathways of toxicity are not clear. The clinical presentation may vary but TMA is a potentially life-threatening condition usually demanding aggressive treatment. We present the case of TMA in a child after living-related liver transplantation (LRLTx) on tacrolimus-based immunosuppressive regiment successfully converted to sirolimus.

Calcium and Electrical Signalling along Endothelium of the Resistance Vasculature

This MiniReview is focused on the nature of intercellular signalling along the endothelium that helps to co-ordinate blood flow control in vascular resistance networks. Vasodilation initiated by contracting skeletal muscle ascends from arterioles within the tissue to encompass resistance arteries upstream and thereby increase blood flow during exercise. In resistance vessels, acetylcholine microiontophoresis or intracellular current injection initiates hyperpolarization that conducts through gap junction channels (GJCs) along the vessel wall resulting in conducted vasodilation (CVD). Both ascending vasodilation and CVD are eliminated with endothelial cell (EC) disruption, pointing to common signalling events and mutual dependence upon EC integrity. As demonstrated by electrical coupling and dye transfer during intracellular recording, their longitudinal orientation and robust expression of GJCs enable ECs to play a predominant role in CVD. Once conduction is initiated, a major interest centres on whether CVD is purely passive or involves additional 'active' signalling events. Here, we discuss components for Ca²⁺ and electrical signalling with an emphasis on intercellular coupling through endothelial GJCs. We stress the importance of understanding relationships between intracellular Ca²⁺ dynamics, EC hyperpolarization and CVD while integrating findings from isolated ECs into more complex interactions in vivo. Whereas endothelial dysfunction accompanies cardiovascular disease and the components of intra- and inter-cellular signalling are increasingly well defined, little is known of how Ca²⁺ signalling and electrical conduction along microvascular endothelium are altered in diseased states. Thus, greater insight into how these relationships are governed and interact is a key goal for continued research efforts.

Studies on the cell biology of interendothelial cell gaps.

Pain, redness, heat, and swelling are hallmarks of inflammation that were recognized as early as the first century AD. Despite these early observations, the mechanisms responsible for swelling, in particular, remained an enigma for nearly two millennia. Only in the past century have scientists and physicians gained an appreciation for the role that vascular endothelium plays in controlling the exudation that is responsible for swelling. One of these mechanisms is the formation of transient gaps between adjacent endothelial cell borders. Inflammatory mediators act on endothelium to reorganize the cytoskeleton, decrease the strength of proteins that connect cells together, and induce transient gaps between endothelial cells. These gaps form a paracellular route responsible for exudation. The discovery that interendothelial cell gaps are causally linked to exudation began in the 1960s and was accompanied by significant controversy. Today, the role of gap formation in tissue edema is accepted by many, and significant scientific effort is dedicated toward developing therapeutic strategies that will prevent or reverse the endothelial cell gaps that are present during the course of inflammatory illness. Given the importance of this field in endothelial cell biology and inflammatory disease, this focused review catalogs key historical advances that contributed to our modern-day understanding of the cell biology of interendothelial gap formation.

Donor Simvastatin Treatment Abolishes Rat Cardiac Allograft Ischemia/Reperfusion Injury and Chronic Rejection Through Microvascular Protection

Ischemia/reperfusion injury may have deleterious short- and long-term consequences for cardiac allografts. The underlying mechanisms involve microvascular dysfunction that may culminate in primary graft failure or untreatable chronic rejection. Here, we report that rat cardiac allograft ischemia/reperfusion injury resulted in profound microvascular dysfunction that was prevented by donor treatment with peroral single-dose simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase and Rho GTPase inhibitor, 2 hours before graft procurement. During allograft preservation, donor simvastatin treatment inhibited microvascular endothelial cell and pericyte RhoA/Rho-associated protein kinase activation and endothelial cell-endothelial cell gap formation; decreased intragraft mRNA levels of hypoxia-inducible factor-1α, inducible nitric oxide synthase, and endothelin-1; and increased heme oxygenase-1. Donor, but not recipient, simvastatin treatment prevented ischemia/reperfusion injury-induced vascular leakage, leukocyte infiltration, the no-reflow phenomenon, and myocardial injury. The beneficial effects of simvastatin on vascular stability and the no-reflow phenomenon were abolished by concomitant nitric oxide synthase inhibition with N-nitro-l-arginine methyl ester and RhoA activation by geranylgeranyl pyrophosphate supplementation, respectively. In the chronic rejection model, donor simvastatin treatment inhibited cardiac allograft inflammation, transforming growth factor-β1 signaling, and myocardial fibrosis. In vitro, simvastatin inhibited transforming growth factor-β1-induced microvascular endothelial-to-mesenchymal transition. Our results demonstrate that donor simvastatin treatment prevents microvascular endothelial cell and pericyte dysfunction, ischemia/reperfusion injury, and chronic rejection and suggest a novel, clinically feasible strategy to protect cardiac allografts.

Leukocyte rolling and adhesion via ICAM-1 signals to endothelial permeability. Focus on “Leukocyte rolling and adhesion both contribute to regulation of microvascular permeability to albumin via ligation of ICAM-1”

THE VASCULAR ENDOTHELIUM plays a vital role in the inflammatory response by induction and surface expression of adhesion molecules and chemokines. At sites of acute inflammation, blood flow is increased and postcapillary venules exhibit increased permeability (leakiness) and support the influx of blood leukocytes, primarily neutrophils. The relationship between neutrophil influx and increased vessel permeability has been a topic of investigation for decades. Many investigations have reported a direct neutrophil-dependent increase in vessel permeability during inflammation using in vivo and in vitro models (reviewed in Ref. 4). One aspect that has received increased attention is whether endothelial cell adhesion molecules contribute to the regulation of vessel permeability, in addition to their well-characterized role in neutrophil rolling and adhesion. Intercellular adhesion molecule-1 (ICAM-1, also known as CD54) is an immunoglobulin superfamily member that is abundantly expressed on the endothelial cell surface and is enriched at endothelial cell borders in vivo and in vitro (2, 12, 14, 17). ICAM-1 surface expression is upregulated in endothelial cells by proinflammatory cytokines like TNF-, IL-1, IFN-, or bacterial endotoxins, and ICAM-1 serves as a receptor for leukocyte 2-integrins (LFA-1 and Mac-1) (reviewed in Ref. 15). In an experiment of nature, patients with leukocyte adhesion deficiency-1 (LAD-1) have a severe primary immune deficiency in which blood neutrophils fail to localize to sites of inflammation or injury. This defect occurs because patients’ neutrophils either lack 2-integrins or contain mutations in these molecules. Studies using neutrophils from LAD-1 patients or 2-integrin-deficient mice clearly demonstrate that these cells have normal selectin-dependent rolling on activated endothelium but fail to stably arrest and transmigrate. Additional studies have demonstrated that endothelial-expressed ICAM-1 contributes to both kinetics of leukocyte rolling and arrest in vivo as determined by intravital microscopy of the cremaster muscle microcirculation in ICAM-1 / mice (16). In this issue of American Journal of Physiology-Cell Physiology, Sumagin and colleagues (18) use elegant in vivo studies to demonstrate that microvascular permeability in the cremaster muscle model is regulated by leukocyte engagement of ICAM-1. The authors monitored permeability (Ps) in cremaster microcirculation by efflux of luminal fluorescent-tagged albumin by fluorescence confocal intravital microscopy. Prior studies by Sarelius and colleagues (19) in the same model demonstrated that ICAM-1 engagement induced a localized increase in vessel permeability in unstimulated arterioles and in TNF-stimulated venules. In agreement with this finding ,4ho f TNF- stimulation of arterioles and venules in ICAM-1 knockout (KO) animals did not result in increased vessel permeability as compared with wild-type (WT) mice under identical conditions. Similar results were seen with 2-integrin KO mice. In the current study, the authors provide further evidence linking ICAM-1 expression to regulation of endothelial permeability. Comparisons between unstimulated venules and TNF--treated venules reveal that neutrophil rolling regulates venule permeability in unstimulated venules while leukocyte adhesion regulates venule permeability in TNF--stimulated venules. The 2-integrin-blocking antibodies, which inhibit neutrophil arrest on endothelium, reduced the vessel permeability, and this was further reduced by treatment with a combination of 2-integrin and P-selectin blocking antibodies that inhibits both rolling and arrest. Interestingly, the reduction in venule permeability observed by blockade of P-selectin and 2-integrin function were suggested to be due to insufficient ICAM-1 engagement due to the absence of rolling leukocytes. To examine the role that neutrophils play, circulating neutrophils were depleted by antineutrophil GR-1 antibody treatment. Notably, depletion of neutrophils caused a marked decrease in

MO‐G‐BRC‐04: Normal Liver Tissue Density Dose‐Response in Patients Treated with Stereotactic Body Radiation Therapy for Liver Metastases

Purpose: To investigate normal liver tissue density changes as a function of dose and time for patients with metastatic liver tumors treated with stereotactic body radiation therapy (SBRT). Methods: Seventy‐one non‐ contrast follow‐up computed‐tomography (CT) scans of 18 patients with liver metastases were retrospectively analyzed from the time of treatment to their final follow‐up scan [range, 1–24 months] [median, 8.2 months]. All patients received a prescription dose of 40–60 Gy [median, 57 Gy] in 3–5 fractions delivered with 3DCRT or dynamic conformal arcs. The follow‐up scans were binned into the following time periods: 3, 6, 9, 12, 18, and 24 months. The liver volume was contoured on all scans for each patient and registered to the planning CT. Dose‐response curves (DRC) were calculated by averaging Hounsfield unit changes (HU) in liver regions (excluding PTV) receiving the same dose in 5 Gy intervals. Results: For early follow‐ ups, a small decrease in normal liver CT numbers (up to −10 HU) was observed from the first to the second follow‐up periods (3 to 6 months); however, this decrease was not observed from treatment to the first follow‐ up period. For later follow‐ups (9, 12, and 18 months), a significant increase in liver CT numbers was seen [up to 20 HU]. A return to the baseline planning CT values was observed for the last follow‐up period (24 months), but only within lower dose regions [&lt; 25 Gy]. Conclusions: The early pathologic veno‐occlusive disease response [Olsen et al., IJROBP 2009] in normal liver after SBRT produces minimal quantitative density change despite its characteristic hypodense CT appearance. The subsequent tissue regenerative process involves transiently increased density that appears to consolidate over time. The high dose region tissue changes are possibly triggered by the mechanism of sinusoidal endothelial cell disruption described in preclinical models [Yamanouchi et al., Hepatology 2009].

A novel vascular disrupting agent plinabulin triggers JNK-mediated apoptosis and inhibits angiogenesis in multiple myeloma cells

AbstractPrevious studies have established a role of vascular-disrupting agents as anti- cancer agents. Plinabulin is a novel vascular-disrupting agent that exhibits potent interruption of tumor blood flow because of the disruption of tumor vascular endothelial cells, resulting in tumor necrosis. In addition, plinabulin exerts a direct action on tumor cells, resulting in apoptosis. In the present study, we examined the anti–multiple myeloma (MM) activity of plinabulin. We show that low concentrations of plinabulin exhibit a potent antiangiogenic action on vascular endothelial cells. Importantly, plinabulin also induces apoptotic cell death in MM cell lines and tumor cells from patients with MM, associated with mitotic growth arrest. Plinabulin-induced apoptosis is mediated through activation of caspase-3, caspase-8, caspase-9, and poly(ADP-ribose) polymerase cleavage. Moreover, plinabulin triggered phosphorylation of stress response protein JNK, as a primary target, whereas blockade of JNK with a biochemical inhibitor or small interfering RNA strategy abrogated plinabulin-induced mitotic block or MM cell death. Finally, in vivo studies show that plinabulin was well tolerated and significantly inhibited tumor growth and prolonged survival in a human MM.1S plasmacytoma murine xenograft model. Our study therefore provides the rationale for clinical evaluation of plinabulin to improve patient outcome in MM.

Contribution of Lethal Toxin and Edema Toxin to the Pathogenesis of Anthrax Meningitis

Bacillus anthracis is a Gram-positive spore-forming bacterium that causes anthrax disease in humans and animals. Systemic infection is characterized by septicemia, toxemia, and meningitis, the main neurological complication associated with high mortality. We have shown previously that B. anthracis Sterne is capable of blood-brain barrier (BBB) penetration, establishing the classic signs of meningitis, and that infection is dependent on the expression of both major anthrax toxins, lethal toxin (LT) and edema toxin (ET). Here we further investigate the contribution of the individual toxins to BBB disruption using isogenic toxin mutants deficient in lethal factor, ΔLF, and edema factor, ΔEF. Acute infection with B. anthracis Sterne and the ΔLF mutant resulted in disruption of human brain microvascular endothelial cell (hBMEC) monolayer integrity and tight junction protein zona occludens-1, while the result for cells infected with the ΔEF mutant was similar to that for the noninfected control. A significant decrease in bacterial invasion of BBB endothelium in vitro was observed during infection with the ΔLF strain, suggesting a prominent role for LT in promoting BBB interaction. Further, treatment of hBMECs with purified LT or chemicals that mimic LT action on host signaling pathways rescued the hypoinvasive phenotype of the ΔLF mutant and resulted in increased bacterial uptake. We also observed that toxin expression reduced bacterial intracellular survival by inducing the bulk degradative autophagy pathway in host cells. Finally, in a murine model of anthrax meningitis, mice infected with the ΔLF mutant exhibited no mortality, brain bacterial load, or evidence of meningitis compared to mice infected with the parental or ΔEF strains.