Rho GTPases mediated integrin α<Subscript>v</Subscript>β<Subscript>3</Subscript> activation in sphingosine-1-phosphate stimulated chemotaxis of endothelial cells

Integrins, a family of transmembrane heterodimeric polypeptides, mediate various biological responses including cell adhesion and migration. In this report, we show that sphingosine-1-phosphate (S1P) activates integrin α_vβ₃ in endothelial cells (ECs) via the sphingosine-1-phosphate receptor subtype 1 (S1P1)-mediated signaling pathway. S1P treatment results in the activation of integrin α_vβ₃ in the lamellipodia region of ECs, suggesting that integrin α_vβ₃ plays a critical role in the S1P-stimulated chemotactic response of ECs. Indeed, S1P treatment induces the association of focal adhesion kinase (FAK) and cytoskeletal proteins with integrin α_vβ₃, the ligation of α_v and β₃ subunits, as well as enhances endothelial migration on vitronectin-coated substrata. Knockdown endothelial S1P1 receptor, treatments with pertussis toxin or dominant-negative-Rho family GTPases abrogates the S1P-induced integrin α_vβ₃ activation in ECs. Consequently, these treatments markedly inhibit the S1P-induced endothelial migratory response on vitronectin-coated substrata. Collectively, these data indicate that the S1P-mediated signaling via the S1P1/G_i/Rho GTPases pathway activates integrin α_vβ₃, which is indispensable for S1P-stimulated chemotactic response of ECs.     

Ezrin/radixin/moesin proteins are phosphorylated by TNF-alpha and modulate permeability increases in human pulmonary microvascular endothelial cells

Endothelial cells (ECs) respond to TNF-alpha by altering their F-actin cytoskeleton and junctional permeability through mechanisms that include protein kinase C (PKC) and p38 MAPK. Ezrin, radixin, and moesin (ERM) regulate many cell processes that often require a conformational change of these proteins as a result of phosphorylation on a conserved threonine residue near the C terminus. This study tested the hypothesis that ERM proteins are phosphorylated on this critical threonine residue through TNF-alpha-induced activation of PKC and p38 and modulate permeability increases in pulmonary microvascular ECs. TNF-alpha induced ERM phosphorylation on the threonine residue that required activation of p38, PKC isoforms, and phosphatidylinositol-4-phosphate 5-kinase Ialpha, a major enzyme generating phosphatidylinositol 4,5-bisphosphate, and phosphorylated ERM were prominently localized at the EC periphery. TNF-alpha-induced ERM phosphorylation was accompanied by cytoskeletal changes, paracellular gap formation, and increased permeability to fluxes of dextran and albumin. These changes required activation of p38 and PKC and were completely prevented by inhibition of ERM protein expression using small interfering RNA. Thus, ERM proteins are phosphorylated through p38 and PKC-dependent mechanisms and modulate TNF-alpha-induced increases in endothelial permeability. Phosphorylation of ERM likely plays important roles in EC responses to TNF-alpha by modulating the F-actin cytoskeleton, adhesion molecules, and signaling events.     

Fibrinogen induces alterations of endothelial cell tight junction proteins

We previously showed that an elevated content of fibrinogen (Fg) increased formation of filamentous actin and enhanced endothelial layer permeability. In the present work we tested the hypothesis that Fg binding to endothelial cells (ECs) alters expression of actin‐associated endothelial tight junction proteins (TJP). Rat cardiac microvascular ECs were grown in gold plated chambers of an electrical cell‐substrate impedance system, 8‐well chambered, or in 12‐well plates. Confluent ECs were treated with Fg (2 or 4 mg/ml), Fg (4 mg/ml) with mitogen‐activated protein kinase (MEK) kinase inhibitors (PD98059 or U0126), Fg (4 mg/ml) with anti‐ICAM‐1 antibody or BQ788 (endothelin type B receptor blocker), endothelin‐1, endothelin‐1 with BQ788, or medium alone for 24 h. Fg induced a dose‐dependent decrease in EC junction integrity as determined by transendothelial electrical resistance (TEER). Western blot analysis and RT‐PCR data showed that the higher dose of Fg decreased the contents of TJPs, occludin, zona occluden‐1 (ZO‐1), and zona occluden‐2 (ZO‐2) in ECs. Fg‐induced decreases in contents of the TJPs were blocked by PD98059, U0126, or anti‐ICAM‐1 antibody. While BQ788 inhibited endothelin‐1‐induced decrease in TEER, it did not affect Fg‐induced decrease in TEER. These data suggest that Fg increases EC layer permeability via the MEK kinase signaling pathway by affecting occludin, ZO‐1, and ZO‐2, TJPs, which are bound to actin filaments. Therefore, increased binding of Fg to its major EC receptor, ICAM‐1, during cardiovascular diseases may increase microvascular permeability by altering the content and possibly subcellular localization of endothelial TJPs. J. Cell. Physiol. 221: 195–203, 2009. © 2009 Wiley‐Liss, Inc     

Homocysteine causes cerebrovascular leakage in mice

Elevated plasma homocysteine (Hcy) is associated with cerebrovascular disease and activates matrix metalloproteinases (MMPs), which lead to vascular remodeling that could disrupt the blood-brain barrier. To determine whether Hcy administration can increase brain microvascular leakage secondary to activation of MMPs, we examined pial venules by intravital video microscopy through a craniotomy in anesthetized mice. Bovine serum albumin labeled with fluorescein isothiocyanate (BSA-FITC) was injected into a carotid artery to measure extravenular leakage. Hcy (30 microM/total blood volume) was injected 10 min after FITC-BSA injection. Four groups of mice were examined: 1) wild type (WT) given vehicle; 2) WT given Hcy (WT + Hcy); 3) MMP-9 gene knockout given Hcy (MMP-9-/- + Hcy); and 4) MMP-9-/- with topical application of histamine (10(-4) M) (MMP-9-/- + histamine). In the WT + Hcy mice, leakage of FITC-BSA from pial venules was significantly (P < 0.05) greater than in the other groups. There was no significant leakage of pial microvessels in MMP-9-/- + Hcy mice. Increased cerebrovascular leakage in the MMP-9-/- + histamine group showed that microvascular permeability could still increase by a mechanism independent of MMP-9. Treatment of cultured mouse microvascular endothelial cells with 30 microM Hcy resulted in significantly greater F-actin formation than in control cells without Hcy. Treatment with a broad-range MMP inhibitor (GM-6001; 1 microM) ameliorated Hcy-induced F-actin formation. These data suggest that Hcy increases microvascular permeability, in part, through MMP-9 activation.     

Actin cytoskeletal isoforms in human endothelial cellsin vitro: Alteration with cell passage

Summary The microfilamentous actin component of the cytoskeleton is crucial to endothelial angiogenesis and vascular permeability. Differences in actin cytoskeletal profiles in cultured human endothelial cells were explored: when first isolated, both primary human umbilical vein endothelial cells (HUVEC) and primary human placental microvascular endothelial cells (HPMEC) expressed F-actin, but notβ-actin orα-smooth muscle actin. A similar endothelial actin profile was observed in cryo-sections of freshly delivered term umbilical cord and placenta. In subsequent cell culture, although the actin cytoskeleton of HUVEC remained unchanged, the actin profiles of HPMEC altered after the second passage with the induction ofα-smooth muscle actin expression, which was intercellularly heterogeneous and increased to 20% at P4. This behavior occurred in HPMEC monolayers cultured on a variety of extracellular matrices. Comparisons with a spontaneously immortalized human microvascular cell-line, HGTEN 21, revealed that inprolonged passage, bothα-smooth muscle actin andβ-actin were expressed, whereas HPMEC at P4 showed a lower level ofβ-actin expression. Therefore, in comparison with large vessels, microvascular cells are more likely to dedifferentiate. This may reflect the ability of microvascular cells to remodel according to changing requirement for new vessel formation. In conclusion, passage of human microvascular endothelial cells, but not of larger vessel endothelial cells, alters the expression of actin isoforms. This may be important in relation to comparisons ofin vitro andin vivo vascular permeability; higher passage microvascular endothelial cells should thus be used with caution in such studies.     

Heterotypic contact reveals a COX-2-mediated suppression of osteoblast differentiation by endothelial cells: A negative modulatory role for prostanoids in VEGF-mediated cell: cell communication?

In bone, angiogenesis must be initiated appropriately, but limited once remodelling or repair is complete. Our recent findings have supported a role for prostaglandins (PG), known modulators of osteoblast (OB) and endothelial cell (EC) behaviour, in facilitating VEGF-mediated paracrine communication from OBs to ‘remotely located’ ECs, but the mechanism(s) regulating OB:EC crosstalk when these cells are closely opposed are undefined. In this study we have examined: (i) the effects of exogenous PGE₂ on VEGF-driven events in ECs, and (ii) the role of endogenous COX-2-derived prostanoids in mediating communication between intimately opposed OBs and ECs in direct contact. Exposure of ECs to PGE₂ increased ERK1/2 phosphorylation, COX-2 induction, 6-keto-PGF_1α release and EC proliferation. In contrast, PGE₂ attenuated VEGF₁₆₅-induced VEGFR2/Flk1 phosphorylation, ERK1/2 activation and proliferation of ECs, suggesting that exogenous PGE₂ restricts the actions of VEGF. However, the COX-2-selective inhibitor, NS398, also attenuated VEGF-induced proliferation, implying a distinct role for endogenous COX-2 activity in regulating EC behaviour. To examine the effect of OB:EC proximity and the role of COX-2 products further, we used a confrontational co-culture model. These studies showed that COX-2 blockade with NS398 enhanced EC-dependent increases in OB differentiation, that this effect was reversed by exogenous PGH₂ (immediate COX-2 product), and that exogenous VEGF did not influence EC-dependent OB differentiation under these conditions. Our findings indicate that locally produced prostanoids may serve distinct roles depending on OB:EC proximity and negatively modulate VEGF-mediated changes in EC behaviour when these cells are closely opposed to control angiogenesis during bone (re)modelling.     

In vitro angiogenesis is modulated by the mechanical properties of fibrin gels and is related to αvβ3 integrin localization

Summary This study deals with the role of the mechanical properties of matrices in in vitro angiogenesis. The ability of rigid fibrinogen matrices with fibrin gels to promote capillarylike structures was compared. The role of the mechanical properties of the fibrin gels was assessed by varying concentration of the fibrin gels. When the concentration of fibrin gels was decreased from 2 mg/ml to 0.5 mg/ml, the capillarylike network increased. On rigid fibrinogen matrices, capillarylike structures were not formed. The extent of the capillarylike network formed on fibrin gels having the lowest concentration depended on the number of cells seeded. The dynamic analysis of capillarylike network formation permitted a direct visualization of a progressive stretching of the 0.5 mg/ml fibrin gels. This stretching was not observed when fibrin concentration increases. This analysis shows that 10 h after seeding, a prearrangement of cells into ringlike structures was observed. These ringlike structures grew in size. Between 16 and 24 h after seeding, the capillarylike structures were formed at the junction of two ringlike structures. Analysis of the α_vβ₃ integrin localization demonstrates that cell adhesion to fibrinogen is mediated through the α_vβ₃ integrin localized into adhesion plaques. Conversely, cell adhesion to fibrin shows a diffuse and dot-contact distribution. We suggest that the balance of the stresses between the tractions exerted by the cells and the resistance of the fibrin gels triggers an angiogenic signal into the intracellular compartment. This signal could be associated with modification in the α_vβ₃ integrin distribution.     

The effect of calcium antagonists on histamine and leukotriene-induced tracheal microvascular permeability in the guine pig

The effects of several calcium antagonists, i.e., nifedipine, verapamil adn 8-[N,N-diethylamino]-octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8), were evaluated on agonist-induced increases in permeability of the airway microvasculature in anesthetized guinea pigs. Vascular permeability was measured as tracheal extravascular albumin content by using ¹²⁵I-bovine serum albumin and the utilization of ⁵¹Cr labelled-erythrocytes to correct for blood volume. Intratracheal injections of histamine (1, 10 and 100 μg) or leukotriene (LT) D₄ (1, 10 and 100 ng) produced dose-dependent increases in extravasated radiolabelled albumin in the trachea. Although histamine produced a greater maximal response than LTD₄, the latter provocation was tent times more potent than the former. Nifedipine, a dihydropyridine calcium slow channel blocker, exhibited dose-dependent (30, 100 and 300 μg/kg) inhibitory activity against histamine-induced increases in extravascular albumin, while another calcium slow channel blocker, verapamil (100, 300 and 1000 μg/kg), exhibited much less activity. TMB-8, a purported intracellular calcium antagonist (1 and 10 mg/kg), was observed to have some inhibitory activity versus histamine. Similar doses of all three calcium antagonists failed to significantly inhibit increases in tracheal microvascular permeability evoked by LTD₄. These results suggest that differences in mediator-induced microvascular permeability in the guinea pig trachea are evident depending upon the agonist selected and the pool of calcium utilized.     

Vascular smooth muscle cells promote endothelial cell adhesion via microtubule dynamics and activation of paxillin and the extracellular signal-regulated kinase (ERK) pathway in a co-culture system

Interaction between endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) plays an important role in vascular biology. Cell adhesion to the extracellular matrix provides critical environmental information necessary for cell migration, proliferation, differentiation and survival. In this study, the role of VSMCs in EC adhesion was demonstrated by using a co-culture system. It was shown that the co-cultured VSMCs significantly increased the number of adherent ECs, and induced an increase of total focal adhesion area in ECs. These changes were associated with a low microtubule-to-tubulin ratio, and activation of extracellular signal-regulated kinase (ERK) and paxillin. Both the EC adhesion state and activation of the ERK/paxillin pathway by the co-cultured VSMCs could be inhibited by trichostatin A (TSA). As an inhibitor of histone deacetylase, TSA acts by modulating microtubule polymerization state. Taken together, these data suggest that the co-cultured VSMCs promote EC adhesion by modulating the microtubule cytoskeleton polymerization state, which in turn activates the ERK pathway and up-regulates phosphorylated paxillin expression to accelerate focal adhesion formation.     

Thyroid hormone receptor α<Subscript>1</Subscript>–β<Subscript>1</Subscript> expression in epididymal epithelium from euthyroid and hypothyroid rats

The objectives of the present work were to assess whether epithelial cells from the different segments of epididymis express TRα₁–β₁ isoforms, to depict its subcellular immunolocalization and to evaluate changes in their expression in rats experimentally submitted to a hypothyroid state by injection of 131I. In euthyroid and hypothyroid groups, TR protein was expressed in epididymal epithelial cells, mainly in the cytoplasmic compartment while only a few one showed a staining in the nucleus as well. A similar TR immunostaining pattern was detected in the different segments of the epididymis. In hypothyroid rats, the number of TR-immunoreactive epithelial cells as well as the intensity of the cytoplasmic staining significantly increased in all sections analyzed. In consonance to the immunocytochemical analysis, the expression of TRα₁–β₁ isoforms, assessed by Western blot revealed significantly higher levels of TR in cytosol compared to the nuclear fractions. Furthermore, TR expression of both α₁ and β₁ isoforms and their mRNA levels were increased by the hypothyroid state. The immuno-electron-microscopy showed specific reaction for TR in principal cells associated with eucromatin, cytosolic matrix and mitochondria. The differences in expression levels assessed in control and thyroidectomized rats ascertain a specific function of TH on this organ.     

β<Subscript>2</Subscript>-Integrin-Mediated Adhesion and Intracellular Ca<Superscript>2+</Superscript> Release in Human Eosinophils

Human eosinophils spontaneously adhere to various substrates in the absence of exogenously added activators. In the present study a method was developed for characterizing eosinophil adhesion by measuring changes in impedance. Impedance measurements were performed in HCO₃-buffered HybriCare medium maintained in a humidified 5% CO₂ incubator at 37°C. Impedance increased by more than 1 kΩ within minutes after eosinophils made contact with the substrate, reaching a peak within 20 min. Blocking mobilization of intracellular [Ca²⁺] that precedes adhesion with BAPTA-AM (10 μM) completely inhibited the rise in impedance as well as the changes in cell shape typically observed in adherent cells. However, lowering the extracellular [Ca²⁺] with 2.5 mM EGTA did not inhibit the increase in impedance. Pretreatment with anti-CD18 antibody to block substrate interactions with β₂-integrins, or jasplakinolide (2 μM) to block actin reorganization, abolished the increase in impedance and adherent morphology of the cells. Exposure of eosinophils to the phosphatidylinositol 3 kinase inhibitor LY294002 (5 μM) or treatment with protein kinase C zeta pseudosubstrate to competitively inhibit activity of the enzyme significantly reduced the increase in impedance and inhibited the cell spreading associated with adhesion. These results demonstrate a novel method for measuring eosinophil adhesion and showed that, following formation of a tethered attachment, a rapid increase in intracellular [Ca²⁺] precedes the cytoskeletal rearrangements required for cell shape changes and plasma membrane-substrate interactions associated with adhesion.     

NEU1 Sialidase Regulates the Sialylation State of CD31 and Disrupts CD31-driven Capillary-like Tube Formation in Human Lung Microvascular Endothelia

The highly sialylated vascular endothelial surface undergoes changes in sialylation upon adopting the migratory/angiogenic phenotype. We recently established endothelial cell (EC) expression of NEU1 sialidase (Cross, A. S., Hyun, S. W., Miranda-Ribera, A., Feng, C., Liu, A., Nguyen, C., Zhang, L., Luzina, I. G., Atamas, S. P., Twaddell, W. S., Guang, W., Lillehoj, E. P., Puché, A. C., Huang, W., Wang, L. X., Passaniti, A., and Goldblum, S. E. (2012) NEU1 and NEU3 sialidase activity expressed in human lung microvascular endothelia. NEU1 restrains endothelial cell migration whereas NEU3 does not. J. Biol. Chem. 287, 15966–15980). We asked whether NEU1 might regulate EC capillary-like tube formation on a Matrigel substrate. In human pulmonary microvascular ECs (HPMECs), prior silencing of NEU1 did not alter tube formation. Infection of HPMECs with increasing multiplicities of infection of an adenovirus encoding for catalytically active WT NEU1 dose-dependently impaired tube formation, whereas overexpression of either a catalytically dead NEU1 mutant, NEU1-G68V, or another human sialidase, NEU3, did not. NEU1 overexpression also diminished EC adhesion to the Matrigel substrate and restrained EC migration in a wounding assay. In HPMECs, the adhesion molecule, CD31, also known as platelet endothelial cell adhesion molecule-1, was sialylated via α2,6-linkages, as shown by Sambucus nigra agglutinin lectin blotting. NEU1 overexpression increased CD31 binding to Arachis hypogaea or peanut agglutinin lectin, indicating CD31 desialylation. In the postconfluent state, when CD31 ectodomains are homophilically engaged, NEU1 was recruited to and desialylated CD31. In postconfluent ECs, CD31 was desialylated compared with subconfluent cells, and prior NEU1 silencing completely protected against CD31 desialylation. Prior CD31 silencing and the use of CD31-null ECs each abrogated the NEU1 inhibitory effect on EC tube formation. Sialyltransferase 6 GAL-I overexpression increased α2,6-linked CD31 sialylation and dose-dependently counteracted NEU1-mediated inhibition of EC tube formation. These combined data indicate that catalytically active NEU1 inhibits in vitro angiogenesis through desialylation of its substrate, CD31.     

串珠素表达下调参与低氧对大鼠心肌微血管内皮细胞增殖的抑制作用

低氧可以抑制内皮细胞增殖,但是其机理目前尚不清楚。串珠素在调节内皮细胞增殖中发挥着重要作用。为了探讨串珠素是否参与低氧对内皮细胞增殖的抑制,将大鼠心肌微血管内皮细胞在低氧或常氧状态下培养12 h后,用实时定量RT-PCR方法检测串珠素mRNA的表达。结果发现：低氧可以明显抑制串珠素mRNA的表达,与常氧状态下串珠素mRNA表达水平比较,差异显著（P〈0．05）。与此同时,低氧状态下或用串珠素抗体中和内源性串珠素,内皮细胞的增殖和对成纤维细胞生长因子的反应明显降低,粘着斑激酶（focal adhesion kinase,FAK）表达和细胞外信号调节激酶1／2（extracellular signal- regulated kinase,ERK1／2）活性明显下降。结果提示,串珠素表达下调可能通过抑制FAK介导的ERK1/2依赖的信号转导途径,参与低氧对大鼠心肌微血管内皮细胞增殖的抑制作用。     

Molecular mechanisms of euplotin C-induced apoptosis: involvement of mitochondrial dysfunction,oxidative stress and proteases

The metabolite euplotin C (EC), isolated from the marine ciliate Euplotes crassus, is a powerful cytotoxic and pro-apoptotic agent in tumour cell lines. For instance, EC induces the rapid depletion of ryanodine Ca²⁺ stores, the release of cytochrome c from the mitochondria, and the activation of caspase-3, leading to apoptosis. The purpose of this study was to gain further insight into the mechanisms of EC-induced apoptosis in rat pheochromocytoma PC12 cells. We found that EC increases Bax/Bcl-2 ratio and that Bax is responsible of the EC-induced dissipation of the mitochondrial membrane potential (Δψ_m). In addition, EC induces the generation of reactive oxygene species (ROS) without involvement of p53. The inhibition of ROS generation prevents, at least in part, the pro-apoptotic effects of EC as well as the effects of EC on Bax, Δψ_m and intracellular free Ca²⁺, indicating a cross-talk between different pathways. However, definition of the effector cascade turns out to be more complex than expected and caspase-independent mechanisms, acting in parallel with caspases, should also be considered. Among them, EC increases the expression/activity of calpains downstream of ROS generation, although calpains seem to exert protective effects. D. Cervia and M. Garcia-Gil equally contributed to the work.     

Roles of M3 receptor in the effect of penehyclidine hydrochloride upregulated beta-arrestin-1 expression in LPS-stimulated HPMVEC

Background: This study is to investigate the roles of muscarinic receptor 3 (M₃ receptor) in the effect of penehyclidine hydrochloride (PHC) upregulated beta-arrestin-1 expression in lipopolysaccharide (LPS)-stimulated human pulmonary microvascular endothelial cell (HPMVEC).

Methods: HPMVECs were transfected with a shRNA-containing plasmid that specifically targets M₃ receptor mRNA. Cells were collected to measure F-actin contents, levels of intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), as well as changes of F-actin cytoskeleton arrangement by Laser scanning confocal. Beta-arrestin-1 protein expressions were determined by Western blot and beta-arrestin-1 mRNA expressions were measured by Real-time PCR.

Results: Similar to normal cells, PHC could also increase F-actin contents and beta-arrestin-1 expressions, reduce ICAM-1 and VCAM-1 expressions, and inhibit LPS-stimulated reorganization of F-actin and formation of stress fiber in M₃ receptor shRNA group. Compared with normal cells, F-actin cytoskeleton was neat, ICAM-1 and VCAM-1 expressions were decreased, as well as F-actin contents were increased in M₃ receptor shRNA group. However, there were no differences in beta-arrestin-1 expressions between normal cell groups and M₃ receptor shRNA groups.

Conclusion: These results indicate that M₃ receptor plays an important role in pulmonary microvascular endothelial barrier function, and knock-out of M₃ receptor could attenuate LPS-induced pulmonary microvascular endothelial injury. However, upregulative effect of PHC on beta-arrestin-1 expression is independent with presence of M₃ receptor.     

Pim protein kinase-3 is regulated by TNF-α and promotes endothelial cell sprouting

Tumor necrosis factor-α (TNF-α) plays an important role in pathological angiogenesis associated with inflammatory response. Pim-3 kinase belonging to serine/threonine protein kinases is a potent suppressor of myc-induced apoptosis. We have recently demonstrated that Pim-3 plays an essential role in endothelial cell (EC) spreading and migration. In this study, we showed that TNF-α transiently increased Pim-3 mRNA expression, and this was mediated through Tumor necrosis factor-α receptor-1 (TNFR1) pathway in ECs. TNF-α could promote stabilization of Pim- 3 mRNA in ECs. Small-interfering RNA (siRNA)-mediated gene knockdown of Pim-3 significantly impaired TNF-α-induced formation of EC membrane protrusions in vitro. Furthermore, Pim-3 silencing inhibited EC sprouting in subcutaneous Matrigel in vivo. eNOS mRNA abundance was lower in Pim-3 siRNA transfected ECs compared with the control ECs. These observations suggest that Pim-3 plays a role in TNF-α-induced angiogenesis.     

Mechanisms regulating GABAergic inhibitory transmission in the basolateral amygdala: implications for epilepsy and anxiety disorders

Summary. The amygdala, a temporal lobe structure that is part of the limbic system, has long been recognized for its central role in emotions and emotional behavior. Pathophysiological alterations in neuronal excitability in the amygdala are characteristic features of certain psychiatric illnesses, such as anxiety disorders and depressive disorders. Furthermore, neuronal excitability in the amygdala, and, in particular, excitability of the basolateral nucleus of the amygdala (BLA) plays a pivotal role in the pathogenesis and symptomatology of temporal lobe epilepsy. Here, we describe two recently discovered mechanisms regulating neuronal excitability in the BLA, by modulating GABAergic inhibitory transmission. One of these mechanisms involves the regulation of GABA release via kainate receptors containing the GluR5 subunit (GluR5KRs). In the rat BLA, GluR5KRs are present on both somatodendritic regions and presynaptic terminals of GABAergic interneurons, and regulate GABA release in an agonist concentration-dependent, bidirectional manner. The relevance of the GluR5KR function to epilepsy is suggested by the findings that GluR5KR agonists can induce epileptic activity, whereas GluR5KR antagonists can prevent it. Further support for an important role of GluR5KRs in epilepsy comes from the findings that antagonism of GluR5KRs is a primary mechanism underlying the antiepileptic properties of the anticonvulsant topiramate. Another mechanism regulating neuronal excitability in the BLA by modulating GABAergic synaptic transmission is the facilitation of GABA release via presynaptic α_1A adrenergic receptors. This mechanism may significantly underlie the antiepileptic properties of norepinephrine. Notably, the α_1A adrenoceptor-mediated facilitation of GABA release is severely impaired by stress. This stress-induced impairment in the noradrenergic facilitation of GABA release in the BLA may underlie the hyperexcitability of the amygdala in certain stress-related affective disorders, and may explain the stress-induced exacerbation of seizure activity in epileptic patients.     

Statin regulated ERK5 stimulates tight junction formation and reduces permeability in human cardiac endothelial cells

The MEKK3/MEK5/ERK5 signaling axis is required for cardiovascular development in vivo. We analyzed the physiological role of ERK5 in cardiac endothelial cells and the consequence of activation of this kinase by the statin class of HMG Co‐A reductase inhibitor drugs. We utilized human cardiac microvascular endothelial cells (HCMECs) and altered ERK5 expression using siRNA mediated gene silencing or overexpression of constitutively active MEK5 and ERK5 to reveal a role for ERK5 in regulating endothelial tight junction formation and cell permeability. Statin treatment of HCMECs stimulated activation of ERK5 and translocation to the plasma membrane resulting in co‐localization with the tight junction protein ZO‐1 and a concomitant reduction in endothelial cell permeability. Statin mediated activation of ERK5 was a consequence of reduced isoprenoid synthesis following HMG Co‐A reductase inhibition. Statin pretreatment could overcome the effect of doxorubicin in reducing endothelial tight junction formation and prevent increased permeability. Our data provide the first evidence for the role of ERK5 in regulating endothelial tight junction formation and endothelial cell permeability. Statin mediated ERK5 activation and the resulting decrease in cardiac endothelial cell permeability may contribute to the cardioprotective effects of statins in reducing doxorubicin‐induced cardiotoxicity.     

Mechanism of action of aflatoxin B1

The inhibitory effects of aflatoxin B₁ were found to be related to the gram character in procaryotes, used in this study. Ethylene diamine tetra chloroacetic acid (0.05% w/v) or Tween-80 (0.05 % v/v) addition accentuated the aflatoxin B₁ growth inhibition inSalmonella typhi andEscherichia coli at different pH values. The inhibition of lipase production was only 5–20 % inPseudomonas fluorescence ca. 25–48% inStaphylococcus aureus andBacillus cereus at different aflatoxin B₁ concentrations (4–16μg/ml).However, inhibition of α-amylase induction was complete in1Bacillus megaterium whereas the inhibition was partial inPseudomonas fluorescence (27–40%) at 32μg aflatoxin B₁ concentration. An increase in leakage of cell contents and decreased inulin uptake were observed in toxin incubated sheep red blood cell suspension (1 %) with increased aflatoxin B₁ concentration