Mitochondria play an important role in 17beta-estradiol attenuation of H(2)O(2)-induced rat endothelial cell apoptosis

Studies have shown salutary effects of 17beta-estradiol following trauma-hemorrhage on different cell types. 17beta-Estradiol also induces improved circulation via relaxation of the aorta and has an anti-apoptotic effect on endothelial cells. Because mitochondria play a pivotal role in apoptosis, we hypothesized that 17beta-estradiol will maintain mitochondrial function and will have protective effects against H(2)O(2)-induced apoptosis in endothelial cells. Endothelial cells were isolated from rats' aorta and cultured in the presence or absence of H(2)O(2), a potent inducer of apoptosis. In additional studies, endothelial cells were pretreated with 17beta-estradiol. Flow cytometry analysis revealed H(2)O(2)-induced apoptosis in 80.9% of endothelial cells; however, prior treatment of endothelial cells with 17beta-estradiol resulted in an approximately 40% reduction in apoptosis. This protective effect of 17beta-estradiol was abrogated when endothelial cells were cultured in the presence ICI-182780, indicating the involvement of estrogen receptor (ER). Fluorescence microscopy revealed a 17beta-estradiol-mediated attenuation of H(2)O(2)-induced mitochondrial condensation. Western blot analysis demonstrated that H(2)O(2)-induced cytochrome c release from mitochondrion to cytosol and the activation of caspase-9 and -3 were decreased by 17beta-estradiol. These findings suggest that 17beta-estradiol attenuated H(2)O(2)-induced apoptosis via ER-dependent activation of caspase-9 and -3 in rat endothelial cells through mitochondria.     

Viral regulation of RANTES expression during human cytomegalovirus infection of endothelial cells

Human cytomegalovirus (HCMV) evades healthy immune responses during infection, and this evasion may allow HCMV to establish latency in the host. The human vasculature has been recognized as a site of HCMV infection and may also be a site of latent HCMV infection. As the interface between circulating cells and underlying parenchymal cells, the vascular endothelium provides signals for local reaction of inflammatory cells. We propose that HCMV down-regulates expression of the proinflammatory chemokine RANTES from the infected endothelium, which may result in reduced recruitment of mononuclear cells to the site of infection. Abortive HCMV infection of primary endothelial cells with the clinical isolate HCMV 4010, under conditions in which viral gene expression could not occur, induced high levels of RANTES expression. Replicative HCMV infection, however, induced cells in parallel cultures to express significantly lower levels of RANTES. Expression of the chemokines interleukin 8 and MCP-1 by endothelial cells was found to be unaffected by replicative HCMV infection and thus may not play an important role during early HCMV infection of the endothelium. HCMV may regulate RANTES expression from endothelial cells as a mechanism to evade the local immune response to infection.     

Suppression of apoptosis by all-trans-retinoic acid. Dual intervention in the c-Jun n-terminal kinase-AP-1 pathway.

Retinoic acid induces apoptosis of various cells, whereas little is known about its anti-apoptotic potential. In this report, we describe an anti-apoptotic property of all-trans-retinoic acid (t-RA) in mammalian cells. Mesangial cells exposed to hydrogen peroxide (H2O2) exhibited shrinkage of the cytoplasm, membrane blebbing, condensation of nuclei, and DNA fragmentation. Pretreatment with t-RA attenuated the morphologic and biochemical hallmarks of apoptosis. t-RA also inhibited apoptosis of mesangial cells triggered by pyrrolidine dithiocarbamate, whereas it did not prevent tumor necrosis factor-alpha-induced apoptosis. The anti-apoptotic effect against H2O2 was similarly observed in NRK49F fibroblasts, but not in Madin-Darby canine kidney epithelial cells and ECV304 endothelial cells. Mesangial cells exposed to H2O2 undergo apoptosis via the activator protein 1 (AP-1)-dependent pathway. We found that t-RA abrogated the H2O2-induced expression of c-fos/c-jun and activation of AP-1. Furthermore, t-RA inhibited H2O2-triggered activation of c-Jun N-terminal kinase (JNK), and dominant-negative inhibition of JNK attenuated the H2O2-induced apoptosis. These data disclosed the novel potential of retinoic acid as an inhibitor of apoptosis. The anti-apoptotic action of t-RA was ascribed, at least in part, to dual suppression of the cell death pathway mediated by JNK and AP-1.     

Human cytomegalovirus (HCMV) infection of endothelial cells promotes naive monocyte extravasation and transfer of productive virus to enhance hematogenous dissemination of HCMV

Human cytomegalovirus (HCMV) pathogenesis is dependent on the hematogenous spread of the virus to host tissue. While data suggest that infected monocytes are required for viral dissemination from the blood to the host organs, infected endothelial cells are also thought to contribute to this key step in viral pathogenesis. We show here that HCMV infection of endothelial cells increased the recruitment and transendothelial migration of monocytes. Infection of endothelial cells promoted the increased surface expression of cell adhesion molecules (intercellular cell adhesion molecule 1, vascular cell adhesion molecule 1, E-selectin, and platelet endothelial cell adhesion molecule 1), which were necessary for the recruitment of na?ve monocytes to the apical surface of the endothelium and for the migration of these monocytes through the endothelial cell layer. As a mechanism to account for the increased monocyte migration, we showed that HCMV infection of endothelial cells increased the permeability of the endothelium. The cellular changes contributing to the increased permeability and increased na?ve monocyte transendothelial migration include the disruption of actin stress fiber formation and the decreased expression of lateral junction proteins (occludin and vascular endothelial cadherin). Finally, we showed that the migrating monocytes were productively infected with the virus, documenting that the virus was transferred to the migrating monocyte during passage through the lateral junctions. Together, our results provide evidence for an active role of the infected endothelium in HCMV dissemination and pathogenesis.     

Low doses of reactive oxygen species protect endothelial cells from apoptosis by increasing thioredoxin-1 expression

The redox regulator thioredoxin-1 (Trx-1) is required for the redox potential of the cell and exerts important functions in cell growth and apoptosis. Severe oxidative stress has been implicated in the oxidation of proteins and cell death. However, the role of low doses of reactive oxygen species (ROS) is poorly understood. Here, we show that 10 and 50 microM H2O2 and short-term exposure to shear stress significantly increased Trx-1 mRNA and protein levels in endothelial cells. Since it is known that Trx-1 exerts anti-apoptotic functions, we next investigated whether low doses of ROS can inhibit basal and serum-depletion induced endothelial cell apoptosis. Indeed, treatment of endothelial cells with 10 and 50 microM H2O2 significantly reduced apoptosis induction. Reduction of Trx-1 expression using an antisense oligonucleotide approach resulted in the induction of apoptosis and abolished the inhibitory effect of low doses of H2O2. Taken together, our results demonstrate that low doses of ROS act as signaling molecules and exert anti-apoptotic functions in endothelial cells via upregulation of the redox-regulator Trx-1.     

Human cytomegalovirus infection of endothelial cells triggers platelet adhesion and aggregation

Human cytomegalovirus (HCMV) is an opportunistic pathogen that has been implicated in the pathogenesis of vascular diseases. HCMV infection of endothelial cells may lead to vascular damage in vitro, and acute-phase HCMV infection has been associated with thrombosis. We hypothesized that viral infection of endothelial cells activates coagulation cascades and contributes to thrombus formation and acute vascular catastrophes in patients with atherosclerotic disease. To assess the effects of HCMV on thrombogenesis, we examined the adhesion and aggregation of blood platelets to uninfected and HCMV-infected endothelial cells. At 7 days after infection, platelet adherence and aggregation were greater in infected than in uninfected cultures (2,000 platelets/100 cells and 225 +/- 15 [mean +/- standard error of the mean] aggregates/five microscopic fields versus 100 platelets/100 cells and no aggregates). von Willebrand factor (vWF), ICAM-1, and VCAM-1 but not collagen IV, E-selectin, P-selectin, CD13, and CD31 were expressed at higher levels on infected cells than on uninfected cells. Platelet aggregation was inhibited by blocking of platelet GPIb (with blocking antibodies) or GPIIb/IIIa (with ReoPro) or by blocking of vWF (with polyclonal antibodies to vWF). Furthermore, blocking of vWF, platelet GPIb, and ICAM-1 but not of the endothelial cell marker CD13, alpha(5)beta(3)-integrin, or HCMV glycoprotein B reduced platelet adherence to infected cells by 75% +/- 5%, 74% +/- 5%, or 18% +/- 5%, respectively. The increased thrombogenicity was dependent on active virus replication and could be inhibited by foscarnet and ganciclovir; these results suggest that a late viral gene may be mediating this phenomenon, which may contribute to vascular catastrophes in patients with atherosclerotic disease.     

Connexin 43 confers resistance to hydrogen peroxide-mediated apoptosis

The current study aimed to understand the anti-apoptotic effect of overexpressed gap junction forming protein connexin (Cx) 43 in C6 glioma cells. C6 cells exposed to hydrogen peroxide (H2O2) or staurosporine demonstrated morphological and biochemical changes consistent with apoptosis, whereas C6 cells expressing Cx43 demonstrated relative resistance to H2O2, but not to staurosporine. This selective protection against H2O2 was due to inhibition of caspase-3 activation in Cx43 expressing cells. siRNA knockdown experiments in rat primary astrocytes confirmed the presence of endogenous Cx43-mediated anti-apoptotic effect. Cx43 interacts with the upstream apoptosis signal-regulating kinase 1 known to mediate H2O2-induced apoptosis providing a possible mechanism for protection. These findings provided new evidence for regulation of the mitogen activated protein kinase pathway and apoptosis by Cx43 implicating this protein in intracellular signaling beyond its role as a gap junction forming protein on the plasma membrane.     

Human cytomegalovirus induces multiple means to combat reactive oxygen species

Reactive oxygen species (ROS) are generated as by-products of many cellular processes and can modulate cellular signaling pathways. However, high ROS levels are toxic; thus, intracellular ROS need to be tightly controlled. Therefore, cells use a group of antioxidant molecules and detoxifying enzymes that remove or detoxify reactive species. We found that the level of the antioxidant glutathione is greatly increased in human cytomegalovirus (HCMV)-infected cells due to activation of glutathione synthetic enzymes. In addition, our data suggest that virus-specific mechanisms are used to induce the expression of target antioxidant and detoxifying enzymes critical for the success of the infection. As a result of this virus-induced anti-ROS environment, key signaling kinases, such as the mammalian target of rapamycin (mTOR) kinase in mTOR complex 1 (mTORC1), are protected from inhibition by exogenous hydrogen peroxide (H(2)O(2)). In this regard, we found that phosphorylation of mTOR kinase at serine 2448 (suggested to be activating) was maintained during infection even under ROS stress conditions that inhibited it in uninfected cells. We also show that AMP-dependent kinase (AMPK)-mediated phosphorylation of serine 792 of raptor, the specificity subunit of mTORC1, increases in infected cells after H(2)O(2) treatment. This phosphorylation is normally inhibitory for mTORC1. However, in infected cells this did not result in inhibition of mTORC1 activity, suggesting that inhibitory effects of raptor phosphorylation are circumvented. Overall, our data suggest that HCMV utilizes virus-specific mechanisms to activate a variety of means to protect the cell and mTORC1 from the effects of ROS.     

Prostacyclin inhibits endothelial cell XIAP ubiquitination and degradation

To understand the role of prostacyclin (PGI(2)) in protecting endothelial cells from apoptosis, we evaluated the effects of carbaprostacyclin (cPGI(2)) on H(2)O(2)-induced human umbilical vein endothelial cell (HUVEC) apoptosis. cPGI(2) suppressed H(2)O(2)-induced annexin V-positive cells in a concentration- and time-dependent manner. Pre-treatment of HUVEC with 50 microM cPGI(2) for 4 h produced the maximal anti-apoptotic effect. Authentic PGI(2) generated by adenoviral transfer of PGI(2) synthetic genes exerted a similar protective effect. cPGI(2) inhibited Smac/DIABLO release from mitochondria, caspase 3 activation, focal adhesion protein degradation, and cell detachment. cPGI(2) selectively protected X-linked inhibitor of apoptosis protein (X-linked IAP, XIAP) from H(2)O(2)-induced ubiquitination, and preserved XIAP protein levels. PD-98059 but not H-89 abrogated the protective action of cPGI(2). cPGI(2) increased ERK phosphorylation which was blocked by PD-98059. HUVEC stably transfected with dominant negative Ras abrogated XIAP preservation by cPGI(2) while constitutive active Ras increased ERK phosphorylation and protected XIAP from degradation. Our results demonstrate for the first time that PGI(2) inhibits XIAP ubiquitination and degradation via the Ras/MEK-1/ERK signaling pathway. Preservation of XIAP proteins represents a key mechanism by which PGI(2) protects endothelial cells from oxidant-induced apoptosis.     

Inhibition of IKKα by BAY61-3606 Reveals IKKα-Dependent Histone H3 Phosphorylation in Human Cytomegalovirus Infected Cells

Protein kinase inhibitors can be used as tools to identify proteins and pathways required for virus replication. Using virus replication assays and western blotting we found that the widely used protein kinase inhibitor BAY61-3606 inhibits replication of human cytomegalovirus (HCMV) strain AD169 and the accumulation of HCMV immediate-early proteins in AD169 infected cells, but has no effect on replication of HCMV strain Merlin. Using in vitro kinase assays we found that BAY61-3606 is a potent inhibitor of the cellular kinase IKKα. Infection of cells treated with siRNA targeting IKKα indicated IKKα was required for efficient AD169 replication and immediate-early protein production. We hypothesized that IKKα was required for AD169 immediate-early protein production as part of the canonical NF-κB signaling pathway. However, although BAY61-3606 inhibited phosphorylation of the IKKα substrate IκBα, we found no canonical or non-canonical NF-κB signaling in AD169 infected cells. Rather, we observed that treatment of cells with BAY61-3606 or siRNA targeting IKKα decreased phosphorylation of histone H3 at serine 10 (H3S10p) in western blotting assays. Furthermore, we found treatment of cells with BAY61-3606, but not siRNA targeting IKKα, inhibited the accumulation of histone H3 acetylation (H3K9ac, H3K18ac and H3K27ac) and tri-methylation (H3K27me3 and H3K36me3) modifications. Therefore, the requirement for IKKα in HCMV replication was strain-dependent and during replication of an HCMV strain requiring IKKα, IKKα-dependent H3S10 phosphorylation was associated with efficient HCMV replication and immediate-early protein production. Plus, inhibition of HCMV replication by BAY61-3606 is associated with acetylation and tri-methylation modifications of histone H3 that do not involve IKKα.     

Cathepsin D and H2O2 stimulate degradation of thioredoxin-1: implication for endothelial cell apoptosis

Cathepsin D (CatD) is a lysosomal aspartic proteinase and plays an important role in the degradation of proteins and in apoptotic processes induced by oxidative stress, cytokines, and aging. All of these stimuli are potent inducers of endothelial cell apoptosis. Therefore, we investigated the role of CatD in endothelial cell apoptosis and determined the underlying mechanisms. Incubation with 100-500 microm H2O2 for 12 h induced apoptosis in endothelial cells. To determine a role for CatD, we co-incubated endothelial cells with the CatD inhibitor pepstatin A. Pepstatin A as well as genetic knock down of CatD abolished H2O2-induced apoptosis. In contrast, overexpression of CatD wild type but not a catalytically inactive mutant of CatD (CatDD295N) induced apoptosis under basal conditions. To gain insights into the underlying mechanisms, we investigated the effect of CatD on reactive oxygen species (ROS) formation. Indeed, knocking down CatD expression reduced H2O2-induced ROS formation and apoptosis. The major redox regulator in endothelial cells is thioredoxin-1 (Trx), which plays a crucial role in apoptosis inhibition. Thus, we hypothesized that CatD may alter Trx protein levels and thereby promote formation of ROS and apoptosis. Incubation with 100 microm H2O2 for 6 h decreased Trx protein levels, whereas Trx mRNA was not altered. H2O2-induced Trx degradation was inhibited by pepstatin A and genetic knock down of CatD but not by other protease inhibitors. Incubation of unstimulated cell lysates with recombinant CatD significantly reduced Trx protein levels in vitro, which was completely blocked by pepstatin A pre-incubation. Overexpression of CatD reduced Trx protein in cells. Moreover, H2O2 incubation led to a translocation of Trx to the lysosomes prior to the induction of apoptosis. Taken together, CatD induces apoptosis via degradation of Trx protein, which is an essential anti-apoptotic and reactive oxygen species scavenging protein in endothelial cells.     

重组nkx2.5腺病毒抑制过氧化氢诱导的H9c2细胞凋亡

为研究心脏发育关键基因nkx2.5的功能及应用价值,构建Ad-Nkx2.5重组腺病毒,并检测nkx2.5过表达拮抗氧化应激损伤的效应及机制。采用AdEasy腺病毒表达系统构建Ad-Nkx2.5重组腺病毒,建立H2O2诱导H9c2心肌细胞凋亡模型,分别用Ad-Nkx2.5重组病毒或对照病毒感染细胞,采用Hoechst33342染色观察细胞形态变化、MTT法检测细胞存活率,免疫印迹检测caspase-3活化、细胞色素C的胞浆含量。并通过Real-timePCR检测凋亡相关基因bcl-2和bax表达。结果发现,nkx2.5过表达促进H9c2细胞存活,抑制H2O2诱导的caspase-3活化及线粒体细胞色素C的释放。Nkx2.5过表达上调bcl-2表达,显著下调H2O2诱导的bax表达。并发现H2O2对Nkx2.5核定位无明显影响。结果显示重组腺病毒介导的Nkx2.5过表达可通过调控凋亡相关基因表达,抑制线粒体凋亡途径,保护心肌细胞抗氧化损伤。     

NAD(P)H oxidase-derived hydrogen peroxide mediates endothelial nitric oxide production in response to angiotensin II

Recently, it has been shown that the exogenous addition of hydrogen peroxide (H(2)O(2)) increases endothelial nitric oxide (NO(.)) production. The current study is designed to determine whether endogenous levels of H(2)O(2) are ever sufficient to stimulate NO(.) production in intact endothelial cells. NO(.) production was detected by a NO(.)-specific microelectrode or by an electron spin resonance spectroscopy using Fe(2+)-(DETC)(2) as a NO(.)-specific spin trap. The addition of H(2)O(2) to bovine aortic endothelial cells caused a potent and dose-dependent increase in NO(.) release. Incubation with angiotensin II (10(-7) mol) elevated intracellular H(2)O(2) levels, which were attenuated with PEG-catalase. Angiotensin II increased NO(.) production by 2-fold, and this was prevented by Losartan and by PEG-catalase, suggesting a critical role of AT1 receptor and H(2)O(2) in this response(.) In contrast, NO(.) production evoked by either bradykinin or calcium ionophore was unaffected by PEG-catalase. As in bovine aortic endothelial cells, angiotensin II doubled NO(.) production in aortic endothelial cells from C57BL/6 mice but had no effect on NO(.) production in endothelial cells from p47(phox-/-) mice. In contrast, stimulated NO(.) production to a similar extent in endothelial cells from wild-type and p47(phox-/-) mice. In summary, the present study provides direct evidence that endogenous H(2)O(2), derived from the NAD(P)H oxidase, mediates endothelial NO(.) production in response to angiotensin II. Under disease conditions associated with elevated levels of angiotensin II, this response may represent a compensatory mechanism. Because angiotensin II also stimulates O(2)() production from the NAD(P)H oxidase, the H(2)O(2) stimulation of NO(.) may facilitate peroxynitrite formation in response to this octapeptide.     

Anti-apoptotic activity of caffeic acid, ellagic acid and ferulic acid in normal human peripheral blood mononuclear cells: a Bcl-2 independent mechanism

Polyphenols have been shown to induce apoptosis in a variety of tumor cells including leukemia both in vitro and in vivo. However, their action on normal human peripheral blood mononuclear cells (PBMCs) during oxidative stress remains to be explored. In this study, we have evaluated the anti-apoptotic and radical scavenging activities of dietary phenolics, namely caffeic acid (CA), ellagic acid (EA) and ferulic acid (FA). H2O2-induced apoptosis in normal human PBMCs was assayed by phosphotidylserine externalization, nucleosomal damage and DNA fragmentation. Incubation of PBMCs with 5 mM H2O2 led to increased Annexin-V binding to externalized phosphatidyl serine (PS), an event of pre-apoptotic stage of the cell. Peripheral blood mononuclear cells pretreated with phenolics could resist H2O2-induced apoptotic damage. Caffeic acid (60 and 120 microM) and EA (100 and 200 microM) caused no change in externalization of PS, whereas FA (100 and 200 microM) increased externalization of PS in PBMCs treated with H2O2. The effects of phenolics were abolished to a large extent by culturing the PBMCs for 24 h after washing the phenolics from the medium. Inhibitory activities of these phenolics on lipid peroxidation were in the order of EA相似文献   

Protection of human endothelial cells from oxidant injury by adenovirus-mediated transfer of the human catalase cDNA.

In a variety of disorders, endothelial cells are exposed to high levels of oxidants, generated within the cells and/or consequent to local inflammation. In the context of the sensitivity of endothelial cells to oxidant stress, particularly related to H2O2, we have designed a replication deficient recombinant adenovirus containing the human catalase cDNA (AdCL) to transfer the catalase cDNA to the endothelial cells, in order to augment intracellular anti-H2O2 protection. Human umbilical vein endothelial cells that were not infected or infected with control adenovirus maintained low levels of catalase mRNA. Endothelial cells infected with AdCL expressed AdCL-driven exogenous catalase mRNA, as early as 24 hr and at least for 7 days. Catalase protein levels were increased significantly over controls in cells infected with AdCL, as were catalase activity levels, with catalase activity correlated closely with levels of catalase protein. Importantly, when the endothelial cells were exposed to 500 microM H2O2, all the AdCL infected endothelial cells survived, compared to only 37% of the control cells. Thus, a recombinant adenovirus containing the human catalase cDNA is able to infect human endothelial cells in vitro and express high levels of functional intracellular catalase, protecting the cells against H2O2-mediated oxidant stress. These observations support the feasibility of the transfer of catalase cDNA to human endothelium to protect against oxidant injury.     

Roles of phosphatidylinositol 3-kinase and NF-kappaB in human cytomegalovirus-mediated monocyte diapedesis and adhesion: strategy for viral persistence

Infected peripheral blood monocytes are proposed to play a key role in the hematogenous dissemination of human cytomegalovirus (HCMV) to tissues, a critical step in the establishment of HCMV persistence and the development of HCMV-associated diseases. We recently provided evidence for a unique strategy involved in viral dissemination: HCMV infection of primary human monocytes promotes their transendothelial migration and differentiation into proinflammatory macrophages permissive for the replication of the original input virus. To decipher the mechanism of hematogenous spread, we focused on the viral dysregulation of early cellular processes involved in transendothelial migration. Here, we present evidence that both phosphatidylinositol 3-kinase [PI(3)K] and NF-kappaB activities were crucial for the HCMV induction of monocyte motility and firm adhesion to endothelial cells. We found that the beta(1) integrins, the beta(2) integrins, intracellular adhesion molecule 1 (ICAM-1), and ICAM-3 were upregulated following HCMV infection and that they played a key role in the firm adhesion of infected monocytes to the endothelium. The viral regulation of adhesion molecule expression is complex, with PI(3)K and NF-kappaB affecting the expression of each adhesion molecule at different stages of the expression cascade. Our data demonstrate key roles for PI(3)K and NF-kappaB signaling in the HCMV-induced cellular changes in monocytes and identify the biological rationale for the activation of these pathways in infected monocytes, which together suggest a mechanism for how HCMV promotes viral spread to and persistence within host organs.     

Role of protein kinase G in barrier-protective effects of cGMP in human pulmonary artery endothelial cells

Increases in endothelial cGMP prevent oxidant-mediated endothelial barrier dysfunction, but the downstream mechanisms remain unclear. To determine the role of cGMP-dependent protein kinase (PKG)(I), human pulmonary artery endothelial cells (HPAEC) lacking PKG(I) expression were infected with a recombinant adenovirus encoding PKG(Ibeta) (Ad.PKG) and compared with uninfected and control-infected (Ad.betagal) HPAEC. Transendothelial electrical resistance (TER), an index of permeability, was measured after H(2)O(2) (250 microM) exposure with or without pretreatment with 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate (CPT-cGMP). HPAEC infected with Ad.PKG, but not Ad.betagal, expressed PKG(I) protein and demonstrated Ser(239) and Ser(157) phosphorylation of vasodilator-stimulated phosphoprotein after treatment with CPT-cGMP. Adenoviral infection decreased basal permeability equally in Ad.PKG- and Ad.betagal-infected HPAEC compared with uninfected cells. Treatment with CPT-cGMP (100 microM) caused a PKG(I)-independent decrease in permeability (8.2 +/- 0.6%). In all three groups, H(2)O(2) (250 microM) caused a similar approximately 35% increase in permeability associated with increased actin stress fiber formation, intercellular gaps, loss of membrane VE-cadherin, and increased intracellular Ca(2+) concentration ([Ca(2+)](i)). In uninfected and Ad.betagal-infected HPAEC, pretreatment with CPT-cGMP (100 microM) partially blocked the increased permeability induced by H(2)O(2). In Ad.PKG-infected HPAEC, CPT-cGMP (50 microM) prevented the H(2)O(2)-induced TER decrease, cytoskeletal rearrangement, and loss of junctional VE-cadherin. CPT-cGMP attenuated the peak [Ca(2+)](i) caused by H(2)O(2) similarly (23%) in Ad.betagal- and Ad.PKG-infected HPAEC, indicating a PKG(I)-independent effect. These data suggest that cGMP decreased HPAEC basal permeability by a PKG(I)-independent process, whereas the ability of cGMP to prevent H(2)O(2)-induced barrier dysfunction was predominantly mediated by PKG(I) through a Ca(2+)-independent mechanism.     

Human cytomegalovirus-induced upregulation of intercellular cell adhesion molecule-1 on villous syncytiotrophoblasts

Human cytomegalovirus (HCMV) is secreted apically from villous trophoblasts, thus congenital infection is not likely to occur by basal release across the basement membrane. As an alternative route, we hypothesize that an HCMV-infected villous syncytiotrophoblast (ST) upregulates intercellular adhesion molecule (ICAM)-1, causing blood monocytes to bind to the ST and induce apoptosis. Purified (>99.99%) populations of human villous trophoblasts were differentiated into an ST-like culture, infected with HCMV strain AD169, and assessed for ICAM-1 expression by immunofluorescence. Infection strongly upregulated ICAM-1 24 h after challenge. ICAM-1 was also stimulated by transfection with viral genes IE2-55, IE1-72, and IE2-86, but not by UV-inactivated virus. Infection with a green fluorescent protein recombinant virus allowed infection and ICAM-1 expression to be topographically located. We found that ICAM-1 was expressed on both infected and noninfected cells. Furthermore, antibody to tumor necrosis factor (TNF)alpha and, to a lesser extent, interleukin (IL)1 beta inhibited ICAM-1 upregulation on noninfected cells but not on infected cells. We conclude that HCMV IE proteins stimulate ICAM-1 expression on villous trophoblasts by paracrine release of TNF alpha and IL1 beta, as well as by a direct effect on infected cells.     

Human cytomegalovirus UL131-128 genes are indispensable for virus growth in endothelial cells and virus transfer to leukocytes

Human cytomegalovirus (HCMV), a ubiquitous human pathogen, is the leading cause of birth defects and morbidity in immunocompromised patients and a potential trigger for vascular disease. HCMV replicates in vascular endothelial cells and drives leukocyte-mediated viral dissemination through close endothelium- leukocyte interaction. However, the genetic basis of HCMV growth in endothelial cells and transfer to leukocytes is unknown. We show here that the UL131-128 gene locus of HCMV is indispensable for both productive infection of endothelial cells and transmission to leukocytes. The experimental evidence for this is based on both the loss-of-function phenotype in knockout mutants and natural variants and the gain-of-function phenotype by trans-complementation with individual UL131, UL130, and UL128 genes. Our findings suggest that a common mechanism of virus transfer may be involved in both endothelial cell tropism and leukocyte transfer and shed light on a crucial step in the pathogenesis of HCMV infection.