Biomedical subjects

Elizabeth O Harrington

Publications and source records attributed to Elizabeth O Harrington.

11 recordsLinked to original sources

PubMed2006

Transforming growth factor-beta1-induced endothelial barrier dysfunction involves Smad2-dependent p38 activation and subsequent RhoA activation.

Lung edema due to increased vascular permeability is a hallmark of acute lung injury and acute respiratory distress syndrome. Both p38 and RhoA signaling events are involved in transforming growth factor (TGF)-beta1-increased endothelial permeability; however, the mechanism by which these pathways cooperate is not clear. In this study, we hypothesized that TGF-beta1-induced changes in endothelial monolayer permeability and in p38 and RhoA activation are dependent on Smad2 signaling. We assessed the role of Smad2 in p38 activation and the role of p38 in RhoA activation by TGF-beta1. We found that TGF-beta1 caused Smad2 phosphorylation between 0.5 and 1 h of exposure in endothelial cells. Knockdown of Smad2 protein prevented TGF-beta1-induced p38 activation and endothelial barrier dysfunction. Furthermore, TGF-beta1-enhanced RhoA activation was dependent on p38 activation. Inhibition of the RhoA-Rho kinase signaling pathway blunted TGF-beta1-induced adherens junction disruption and focal adhesion complex formation. In addition, depletion of heat shock protein 27, a downstream signaling molecule of p38, did not prevent TGF-beta1-induced endothelial barrier dysfunction. Finally, inhibition of de novo protein expression blunted TGF-beta1-induced RhoA activation and endothelial barrier dysfunction. Our data indicate that TGF-beta1 induces endothelial barrier dysfunction involving Smad2-dependent p38 activation, resulting in RhoA activation by possible transcriptional regulation.

Animals↗

PubMed2006

Natriuretic peptides differentially attenuate thrombin-induced barrier dysfunction in pulmonary microvascular endothelial cells.

Previous studies have described a protective effect of atrial natriuretic peptide (ANP) against agonist-induced permeability in endothelial cells derived from various vascular beds. In the current study, we assessed the effects of the three natriuretic peptides on thrombin-induced barrier dysfunction in rat lung microvascular endothelial cells (LMVEC). Both ANP and brain natriuretic peptide (BNP) attenuated the effect of thrombin on increased endothelial monolayer permeability and significantly enhanced the rate of barrier restoration. C-type natriuretic peptide (CNP) had no effect on the degree of thrombin-induced monolayer permeability, but did enhance the restoration of the endothelial barrier, similar to ANP and BNP. In contrast, the non-guanylyl cyclase-linked natriuretic peptide receptor specific ligand, cyclic-atrial natriuretic factor (c-ANF), delayed the rate of barrier restoration following exposure to thrombin. All three natriuretic peptides promoted cGMP production in the endothelial cells; however, 8-bromo-cGMP alone did not significantly affect thrombin modulation of endothelial barrier function. ANP and BNP, but not CNP or c-ANF, blunted thrombin-induced RhoA GTPase activation. We conclude that ANP and BNP protect against thrombin-induced barrier dysfunction in the pulmonary microcirculation by a cGMP-independent mechanism, possibly by attenuation of RhoA activation.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed2005

PKCdelta regulates endothelial basal barrier function through modulation of RhoA GTPase activity.

We have shown that PKCdelta enhanced microvascular endothelial basal barrier function, correlating with elevated RhoA GTPase activity and increased focal contact formation. In the current study, we investigated signaling pathways important in PKCdelta modulation of barrier function in unstimulated endothelial cell monolayers by assessing the effects of PKCdelta inhibition in endothelial cells (EC) derived from rat pulmonary artery (PAEC) and epididymus (FPEC). Rottlerin exposure or Ad PKCdeltadn infection significantly enhanced monolayer permeability in both EC. Immunofluorescence analyses demonstrated fewer stress fibers and focal contacts in rottlerin-treated or Ad PKCdeltadn-infected EC; yet, PKCdelta inhibition caused no significant changes in microtubule structures. These changes correlated with a reduction in both focal adhesion kinase (FAK) and RhoA GTPase activities. Microfilament stabilization significantly attenuated the focal contact and barrier disruptive effects of rottlerin. FAK overexpression did not blunt the effects of rottlerin-induced barrier dysfunction or stress fiber and focal contact disruption. Conversely, GFP-linked dominant active RhoA overexpression protected EC from stress fiber and focal contact disruption induced by both rottlerin exposure and overexpression of PKCdelta dominant negative protein. Additionally, PKCdelta immunoprecipitated with p190RhoGAP and p120RasGAP, modulators of RhoA activity. Thus, PKCdelta may regulate basal endothelial barrier function by stabilizing microfilaments and focal contacts by regulating RhoA GTPase activity through upstream modulators, p190RhoGAP and p120RasGAP.

Actin Cytoskeleton↗

PubMed2005

Apoptosis and lung injury.

Apoptosis is important in developmental biology and in remodeling of tissues during repair. Apoptosis also plays important roles in the progression of many diseases. The cellular and molecular mechanisms of apoptosis, in general, have been extensively demonstrated. However, the causes and the roles of apoptosis of various cell types in the lung are not well understood. We have determined that adenosine/homocysteine causes lung vascular endothelial cell apoptosis by inhibition of carboxyl methylation of the small GTPase, Ras, through inhibition of isoprenylcysteine carboxyl methyltransferase(ICMT) activity, leading to inactivation of Ras and the subsequent disruption of focal adhesion complexes, resulting in cell-extracellular matrix detachment and anoikis. Apoptosis can either ameliorate or exacerbate lung injury, depending upon the cell type. Although apoptosis of polymorphonuclear leukocytes in the lung prevents inflammation and the development of acute respiratory distress syndrome during acute lung injury, Fas/FasL-mediated alveolar epithelial cell apoptosis promotes acute lung injury and pulmonary fibrosis. Lung epithelial and endothelial cell apoptosis also contributes to the development of emphysema. This article focuses on elucidating the mechanisms of adenosine/homocysteine-induced endothelial cell apoptosis. We also review the current understanding of the role of lung cell apoptosis in acute lung injury, pulmonary fibrosis and emphysema.

Animals↗

PubMed2004

Barrier dysfunction and RhoA activation are blunted by homocysteine and adenosine in pulmonary endothelium.

RhoA GTPases modulate endothelial permeability. We have previously shown that adenosine and homocysteine enhance basal barrier function in pulmonary artery endothelial cells by a mechanism involving diminution of RhoA carboxyl methylation and activity. In the current study, we investigated the effects of adenosine and homocysteine on endothelial monolayer permeability in cultured monolayers. Adenosine and homocysteine significantly attenuated thrombin-induced endothelial barrier dysfunction and intercellular gap formation. We found significantly diminished RhoA associated with the membrane subcellular fraction in endothelial cells pretreated with adenosine and homocysteine, compared with vehicle-treated endothelial cells. Additionally, adenosine and homocysteine significantly blunted RhoA activation following thrombin exposure. Incubation with adenosine and homocysteine also enhanced in vitro interactions between RhoA and RhoGDI, as well as subcellular translocation of p190RhoGAP to the cytosol. These data demonstrate that elevated intracellular concentrations of homocysteine and adenosine enhance endothelial barrier function in cultured endothelial cells isolated from the main pulmonary artery and lung microvasculature, suggesting a potentially protective effect against pulmonary edema in response to lung injury. We speculate that homocysteine and adenosine modulate the level of endothelial barrier dysfunction through modulation of RhoA posttranslational processing resulting in diminished GTPase activity through altered interactions with modulators of RhoA activation.

Adenosine↗

PubMed2004

The lectin-like domain of complement receptor 3 protects endothelial barrier function from activated neutrophils.

The adhesion of neutrophils to endothelial cells is a central event leading to diapedesis and involves the binding of the I-domain of beta(2) integrins (CD11/CD18) to endothelial ICAMs. In addition to the I-domain, the beta(2) integrin complement receptor 3 (CR3) (CD11b/CD18) contains a lectin-like domain (LLD) that can alter leukocyte functions such as chemotaxis and cytotoxicity. The present study demonstrates that, in contrast to the CR3 I-domain, Ab blockade of the CR3 LLD has no role in mediating neutrophil-induced loss of endothelial barrier function. However, activation of CR3 with the LLD agonist beta-glucan protects the barrier function of endothelial cells in the presence of activated neutrophils and reduces transendothelial migration without affecting adhesion of the neutrophils to the endothelium. The LLD site-specific mAb VIM12 obviates beta-glucan protection while activation of the LLD by VIM12 cross-linking mimics the beta-glucan response by both preserving endothelial barrier function and reducing neutrophil transendothelial migration. beta-glucan has no direct effect on endothelial cell function in the absence of activated neutrophils. These findings demonstrate that signaling through the CR3 LLD prevents neutrophil-induced loss of endothelial barrier function and reduces diapedesis. This suggests that the LLD may be a suitable target for oligosaccharide-based anti-inflammatory therapeutics.

Chemotaxis, Leukocyte↗

PubMed2003

Isoprenylcysteine carboxyl methyltransferase modulates endothelial monolayer permeability: involvement of RhoA carboxyl methylation.

RhoA and Rac1 regulate formation of stress fibers and intercellular junctions, thus modulating endothelial monolayer permeability. Posttranslational modifications of RhoA and Rac1 regulate enzyme activity and subcellular localization, resulting in altered cellular function. The role of RhoA and Rac1 carboxyl methylation in modulating endothelial monolayer permeability is not known. In this study, we found that inhibition of isoprenylcysteine-O-carboxyl methyltransferase (ICMT) with adenosine plus homocysteine or N-acetyl-S-geranylgeranyl-l-cysteine decreased RhoA carboxyl methylation, RhoA activity, and endothelial monolayer permeability, suggesting that RhoA carboxyl methylation may play a role in the ICMT-modulated monolayer permeability. Similar studies showed no effect of ICMT inhibition on Rac1 carboxyl methylation or localization. Bovine pulmonary artery endothelial cells (PAECs) stably overexpressing ICMT-GFP cDNA were established to determine if increased ICMT expression could alter RhoA or Rac1 carboxyl methylation, activation, and endothelial monolayer permeability. PAECs stably overexpressing ICMT demonstrated increased RhoA carboxyl methylation, membrane-bound RhoA, and RhoA activity. Additionally, PAECs stably overexpressing ICMT had diminished VE-cadherin and beta-catenin at intercellular junctions, with resultant intercellular gap formation, as well as enhanced monolayer permeability. These effects were blunted by adenosine plus homocysteine and by inhibition of RhoA, but not by inhibition of Rac1. These results indicate that ICMT modulates endothelial monolayer permeability by altering RhoA carboxyl methylation and activation, thus changing the organization of intercellular junctions. Therefore, carboxyl methylation of RhoA may modulate endothelial barrier function.

Adenosine↗

PubMed2003

Isoprenylcysteine carboxyl methyltransferase activity modulates endothelial cell apoptosis.

Extracellular ATP, adenosine (Ado), and adenosine plus homocysteine (Ado/HC) cause apoptosis of cultured pulmonary artery endothelial cells through the enhanced formation of intracellular S-adenosylhomocysteine and disruption of focal adhesion complexes. Because an increased intracellular ratio of S-adenosylhomocysteine/S-adenosylmethionine favors inhibition of methylation, we hypothesized that Ado/HC might act by inhibition of isoprenylcysteine-O-carboxyl methyltransferase (ICMT). We found that N-acetyl-S-geranylgeranyl-L-cysteine (AGGC) and N-acetyl-S-farnesyl-L-cysteine (AFC), which inhibit ICMT by competing with endogenous substrates for methylation, caused apoptosis. Transient overexpression of ICMT inhibited apoptosis caused by Ado/HC, UV light exposure, or tumor necrosis factor-alpha. Because the small GTPase, Ras, is a substrate for ICMT and may modulate apoptosis, we also hypothesized that inhibition of ICMT with Ado/HC or AGGC might cause endothelial apoptosis by altering Ras activation. We found that ICMT inhibition decreased Ras methylation and activity and the activation of the downstream signaling molecules Akt, ERK-1, and ERK-2. Furthermore, overexpression of wild-type or dominant active H-Ras blocked Ado/HC-induced apoptosis. These findings suggest that inhibition of ICMT causes endothelial cell apoptosis by attenuation of Ras GTPase methylation and activation and its downstream antiapoptotic signaling pathway.

Acetylcysteine↗

PubMed2003

Role of protein kinase C isoforms in rat epididymal microvascular endothelial barrier function.

Endothelial barrier dysfunction is involved in a variety of diseased states. We investigated the role of protein kinase C (PKC) in monolayer permeability using endothelial cells (EC) overexpressing PKC alpha (PKC alpha EC), PKC delta (PKC delta EC) or vector (vector control EC) cDNAs. Thrombin induced permeability changes in all EC, and induced significantly elevated rates of monolayer permeability in PKC alpha EC. Conversely, the basal level of permeability was significantly blunted in PKC delta EC, resulting in diminished thrombin-induced changes in permeability. PKC inhibitors, Gö6976 and rottlerin, reversed the effects of PKC alpha and PKC delta overexpression on permeability, respectively. Immunoblot analyses demonstrated significantly less beta-catenin associated with the cytoskeletal subcellular fraction in thrombin-treated PKC alpha EC, an effect blocked by pretreatment with Gö6976. PKC delta EC contained significantly greater numbers of focal contacts per cell. Thrombin enhanced RhoA GTPase activity in all EC; with a 3-fold greater level of activity in PKC delta EC. Rottlerin significantly blunted RhoA GTPase activity in all EC. Overexpression of RhoA dominant-negative cDNA diminished the size and number of focal contacts in EC, and significantly enhanced the basal rate of PKC delta EC monolayer permeability. These findings demonstrate that monolayer permeability changes are differentially regulated by PKC isoenzymes, suggesting that PKC alpha promotes endothelial barrier dysfunction and PKC delta enhances basal endothelial barrier function.

Acetophenones↗

PubMed2002

Conventional protein kinase C mediates phorbol-dibutyrate-induced cytoskeletal remodeling in a7r5 smooth muscle cells.

Phorbol dibutyrate (PDBu) induced the formation of podosome-like structures together with partial disassembly of actin stress fibers in A7r5 smooth muscle cells. These podosomes contained alpha-actinin, F-actin, and vinculin and exhibit a tubular, column-like structure arising perpendicularly from the bottom of PDBu-treated cells. The conventional protein kinase C (PKC) antagonist, GO6976, inhibited PDBu-induced cytoskeletal remodeling at 0.1 microM, whereas the novel PKC antagonist, rottlerin, was ineffective at 10 microM. PDBu induced the translocation of the conventional PKC-alpha but not the novel PKC-delta to the sites of podosome formation in A7r5 cells. Although partial disassembly of actin stress fibers was observed in both Y-27632- and PDBu-treated cells, focal adhesions were much reduced in number and size only in Y-27632-treated cells. Furthermore, PDBu restored focal adhesions in Y-27632-treated cells. Live video fluorescence microscopy of alpha-actinin GFP revealed a lag phase of about 20 min prior to the rapid formation and dynamic reorganization of podosomes during PDBu treatment. These findings suggest that conventional PKCs mediate PDBu-induced formation of dynamic podosome-like structures in A7r5 cells, and Rho-kinase is unlikely to be the underlying mechanism. The podosome columns could represent molecular scaffolds where PKC-alpha phosphorylates regulatory proteins necessary for Ca(2+) sensitization in smooth muscle cells.

Acetophenones↗

PubMed2002

FAK blunts adenosine-homocysteine-induced endothelial cell apoptosis: requirement for PI 3-kinase.

Treatment of cultured bovine pulmonary endothelial cells (BPAEC) with adenosine (Ado) alone or in combination with homocysteine (Hc) leads to disruption of focal adhesion complexes, caspase-dependent degradation of components of focal adhesion complexes, and subsequent apoptosis. Endothelial cells transiently overexpressing paxillin or p130(Cas) cDNAs underwent Ado-Hc-induced apoptosis to an extent similar to that of cells transfected with vector alone. However, overexpression of focal adhesion kinase (FAK) cDNA blunted Ado-Hc-induced apoptosis. FAK constructs lacking the central catalytic domain or containing a point mutation, rendering the catalytic domain enzymatically inactive, did not provide protection from apoptosis. Constructs containing a mutation in the major autophosphorylation site (tyrosine-397) similarly did not prevent cell death. A FAK mutant in amino acid 395, deficient in phosphatidylinositol 3-kinase (PI 3-kinase) binding, was not able to blunt apoptosis. Finally, overexpression of FAK did not provide protection from apoptosis in the presence of LY-294002, a PI 3-kinase inhibitor. Taken together, these data suggest that the survival signals mediated by overexpression of FAK in response to Ado-Hc-induced apoptosis require a PI 3-kinase-dependent pathway.

Adenosine↗