Elizabeth O. Harrington

The Diagnostic Accuracy of Ex Vivo MRI for Human Atherosclerotic Plaque Characterization

Recent evidence indicates that the type of atherosclerotic plaque, rather than the degree of obstruction to flow, is an important determinant of the risk of cardiovascular complications. In previous work, the feasibility of using MRI for the characterization of plaque components was shown. This study extends the previous work to all the plaque components and shows the accuracy of this method. Twenty-two human carotid endarterectomy specimens underwent ex vivo MRI and histopathological examination. Sixty-six cross sections were matched between MRI and histopathology. In each cross section, the presence or absence of plaque components were prospectively identified on the MRI images. The overall sensitivity and specificity for each tissue component were very high. Calcification and fibrocellular tissue were readily identified. Lipid core was also identifiable. However, thrombus was the plaque component for which MRI had the lowest sensitivity. A semiautomated algorithm was created to identify all major atherosclerotic plaque components. MRI can characterize carotid artery plaques with a high level of sensitivity and specificity. Application of these results in the clinical setting may be feasible in the near future.

Induction of Vascular Permeability by the Sphingosine-1-Phosphate Receptor–2 (S1P2R) and its Downstream Effectors ROCK and PTEN

Objectives—S1P acts via the S1PR family of G protein–coupled receptors to regulate a variety of physiological responses. Whereas S1P1R activates Gi- and PI-3-kinase–dependent signals to inhibit vascular permeability, the related S1P2R inhibits the PI-3-kinase pathway by coupling to the Rho-dependent activation of the PTEN phosphatase. However, cellular consequences of S1P2R signaling in the vascular cells are not well understood. Methods and Results—Selective signaling of the S1P2R was achieved by adenoviral-mediated expression in endothelial cells. Secondly, endogenously expressed S1P2R was blocked by the specific pharmacological antagonist JTE013. Activation of S1P2R in endothelial cells resulted in Rho-ROCK– and PTEN-dependent disruption of adherens junctions, stimulation of stress fibers, and increased paracellular permeability. JTE013 treatment of naive endothelial cells potentiated the S1P1R-dependent effects such as formation of cortical actin, blockade of stress fibers, stimulation of adherens junction assembly, and improved barrier integrity. This observation was extended to the in vivo model of vascular permeability in the rat lung: the S1P2R antagonist JTE013 significantly inhibited H2O2-induced permeability in the rat lung perfused model. Conclusions—S1P2R activation in endothelial cells increases vascular permeability. The balance of S1P1 and S1P2 receptors in the endothelium may determine the regulation of vascular permeability by S1P.

Requirement for Protein Kinase C Activation in Basic Fibroblast Growth Factor–Induced Human Endothelial Cell Proliferation

The intracellular signaling mechanisms that mediate basic fibroblast growth factor (bFGF)-induced angiogenesis have not been fully identified. In particular, whether activation of the intracellular enzyme protein kinase C (PKC) is necessary or sufficient for bFGF-induced mitogenesis of human endothelial cells is not clear. Accordingly, the effect of bFGF stimulation on the Ca2+ increase and PKC activity of normal human endothelial cells (HEC) was studied, as was the effect of inhibition of PKC and the distribution of PKC isoenzymes in these cells. The addition of bFGF to cultured HEC increased overall PKC activity in the absence of an increase in intracellular Ca2+ and markedly stimulated their proliferation, as did the addition of PKC-activating phorbol esters. bFGF-induced proliferation was prevented by the PKC inhibitors chelerythrine and H-7 and by downregulation of PKC after prolonged incubation with phorbol esters. In contrast, these inhibitors did not prevent HEC proliferation induced by epidermal growth factor. Because of the failure of bFGF to increase Ca2+, we determined whether bFGF-induced proliferation could be mediated by novel or atypical PKC isoenzymes (which are not regulated by Ca2+). Investigation of the isoenzyme distribution of confluent and subconfluent HEC by immunoblotting, Northern transfer analysis, and polymerase chain reaction of reverse-transcribed RNA revealed the presence of several novel and atypical isoenzymes (PKC-delta, -eta, -theta, and -zeta) as well as small amounts of the conventional (Ca(2+)-regulated) isoenzymes PKC-alpha and -beta. Activation of PKC by bFGF, in the absence of an increase in intracellular Ca2+, suggests that one or more of these Ca(2+)-independent PKC isoenzymes are both necessary and sufficient for HEC proliferation after bFGF.

Leukocyte Scanning With 111In Is Superior to Magnetic Resonance Imaging in Diagnosis of Clinically Unsuspected Osteomyelitis in Diabetic Foot Ulcers

OBJECTIVE To compare the accuracies of MRI and leukocyte scanning in diagnosing clinically unsuspected osteomyelitis in diabetic foot ulcers. RESEARCH DESIGN AND METHODS A prospective study of 16 diabetic foot ulcers in 12 patients, including both ambulatory and hospitalized patients, was performed at a university medical center. Pedal images were obtained by leukocyte scanning with [111In]oxyquinoline and MRI. Definitive diagnosis of osteomyelitis then was determined by bone biopsy for culture and histology. RESULTS Biopsy-proven osteomyelitis was present in 7 (44%) of the 16 foot ulcers. The diagnosis was suspected clinically in 0%. Leukocyte scanning was 100% sensitive, whereas MRI was only 29% sensitive in diagnosing osteomyelitis in diabetic foot ulcers. Specificities were 67 and 78%, respectively. The positive and negative predictive values (70 and 100%, respectively) for the leukocyte scan also were > those of MRI (50 and 58%, respectively). CONCLUSIONS Leukocyte scanning is superior to MRI in detecting clinically unsuspected osteomyelitis in diabetic foot ulcers.

Role of protein tyrosine phosphatase SHP2 in barrier function of pulmonary endothelium

Pulmonary edema is mediated in part by disruption of interendothelial cell contacts. Protein tyrosine phosphatases (PTP) have been shown to affect both cell-extracellular matrix and cell-cell junctions. The SH2 domain-containing nonreceptor PTP, SHP2, is involved in intercellular signaling through direct interaction with adherens junction proteins. In this study, we examined the role of SHP2 in pulmonary endothelial barrier function. Inhibition of SHP2 promoted edema formation in rat lungs and increased monolayer permeability in cultured lung endothelial cells. In addition, pulmonary endothelial cells demonstrated a decreased level of p190RhoGAP activity following inhibition of SHP2, events that were accompanied by a concomitant increase in RhoA activity. Furthermore, immunofluorescence microscopy confirmed enhanced actin stress fiber formation and diminished interendothelial staining of adherens junction complex-associated proteins upon SHP2 inhibition. Finally, immunoprecipitation and immunoblot analyses demonstrated increased tyrosine phosphorylation of VE-cadherin, beta-catenin, and p190RhoGAP proteins, as well as decreased association between p120-catenin and VE-cadherin proteins. Our findings suggest that SHP2 supports basal pulmonary endothelial barrier function by coordinating the tyrosine phosphorylation profile of VE-cadherin, beta-catenin, and p190RhoGAP and the activity of RhoA, signaling molecules important in adherens junction complex integrity.

Isoprenylcysteine Carboxyl Methyltransferase Modulates Endothelial Monolayer Permeability: Involvement of RhoA Carboxyl Methylation

Abstract— 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 &bgr;-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 protected against pulmonary edema through transporter- and receptor A2-mediated endothelial barrier enhancement

We have previously demonstrated that adenosine plus homocysteine enhanced endothelial basal barrier function and protected against agonist-induced barrier dysfunction in vitro through attenuation of RhoA activation by inhibition of isoprenylcysteine-O-carboxyl methyltransferase. In the current study, we tested the effect of elevated adenosine on pulmonary endothelial barrier function in vitro and in vivo. We noted that adenosine alone dose dependently enhanced endothelial barrier function. While adenosine receptor A(1) or A(3) antagonists were ineffective, an adenosine transporter inhibitor, NBTI, or a combination of DPMX and MRS1754, antagonists for adenosine receptors A(2A) and A(2B), respectively, partially attenuated the barrier-enhancing effect of adenosine. Similarly, inhibition of both A(2A) and A(2B) receptors with siRNA also blunted the effect of adenosine on barrier function. Interestingly, inhibition of both transporters and A(2A)/A(2B) receptors completely abolished adenosine-induced endothelial barrier enhancement. The adenosine receptor A(2A) and A(2B) agonist, NECA, also significantly enhanced endothelial barrier function. These data suggest that both adenosine transporters and A(2A) and A(2B) receptors are necessary for exerting maximal effect of adenosine on barrier enhancement. We also found that adenosine enhanced Rac1 GTPase activity and overexpression of dominant negative Rac1 attenuated adenosine-induced increases in focal adhesion complexes. We further demonstrated that elevation of cellular adenosine by inhibition of adenosine deaminase with Pentostatin significantly enhanced endothelial basal barrier function, an effect that was also associated with enhanced Rac1 GTPase activity and with increased focal adhesion complexes and adherens junctions. Finally, using a non-inflammatory acute lung injury (ALI) model induced by alpha-naphthylthiourea, we found that administration of Pentostatin, which elevated lung adenosine level by 10-fold, not only attenuated the development of edema before ALI but also partially reversed edema after ALI. The data suggest that adenosine deaminase inhibition may be useful in treatment of pulmonary edema in settings of ALI.

Cigarette smoke causes lung vascular barrier dysfunction via oxidative stress-mediated inhibition of RhoA and focal adhesion kinase

Cigarette smoke (CS) is a major cause of chronic lung and cardiovascular diseases. Recent studies indicate that tobacco use is also a risk factor for acute lung injury (ALI) associated with blunt trauma. Increased endothelial cell (EC) permeability is a hallmark of ALI. CS increases EC permeability in vitro and in vivo; however, the underlying mechanism is not well understood. In this study, we found that only 6 h of exposure to CS impaired endothelial barrier function in vivo, an effect associated with increased oxidative stress in the lungs and attenuated by the antioxidant N-acetylcysteine (NAC). CS also exacerbated lipopolysaccharide (LPS)-induced increase in vascular permeability in vivo. Similar additive effects were also seen in cultured lung EC exposed to cigarette smoke extract (CSE) and LPS. We further demonstrated that CSE caused disruption of focal adhesion complexes (FAC), F-actin fibers, and adherens junctions (AJ) and decreased activities of RhoA and focal adhesion kinase (FAK) in cultured lung EC. CSE-induced inhibition of RhoA and FAK, endothelial barrier dysfunction, and disassembly of FAC, F-actin, and AJ were prevented by NAC. In addition, the deleterious effects of CSE on FAC, F-actin fibers, and AJ were blunted by overexpression of constitutively active RhoA and of FAK. Our data indicate that CS causes endothelial barrier dysfunction via oxidative stress-mediated inhibition of RhoA and FAK.

Transforming growth factor-β1 causes pulmonary microvascular endothelial cell apoptosis via ALK5

We have previously shown that transforming growth factor (TGF)-beta1 protected against main pulmonary artery endothelial cell (PAEC) apoptosis induced by serum deprivation and VEGF receptor blockade through a mechanism associated with ALK5-mediated Bcl-2 upregulation. In the current study, we investigated the effect of TGF-beta1 on pulmonary microvascular endothelial cell (PMVEC) apoptosis. We found that, in contrast to the results seen in conduit PAEC, TGF-beta1 caused apoptosis of PMVEC, an effect that was also dependent on ALK5 activity. We noted that non-SMAD signaling pathways did not play a role in TGF-beta1-induced apoptosis. Both SMAD2 and SMAD1/5 were activated upon exposure to TGF-beta1. TGF-beta1-induced activation of SMAD2, but not SMAD1/5, was abolished by ALK5 inhibition, an effect that associated with prevention of TGF-beta1-induced apoptosis. These results suggest that SMAD2 is important in TGF-beta1-induced apoptosis of PMVEC. While caspase-12 activity was not altered, caspase-8 was activated by TGF-beta1, an effect that correlated with a reduction of cFLIP protein levels. Additionally, TGF-beta1 decreased Bcl-2 protein levels and induced cytochrome c cytosolic redistribution. These results suggest that TGF-beta1 caused apoptosis of PMVEC likely through both caspase-8-dependent extrinsic pathway and mitochondria-mediated intrinsic pathway. We noted that inhibition of ALK5 attenuated serum deprivation-induced apoptosis, an effect that correlated with increased expression and activation of CREB and its potential target genes, Bcl-2 and cFLIP. These results suggest that CREB may be important in mediating apoptosis resistance of PMVEC upon ALK5 inhibition perhaps through upregulation of Bcl-2 and cFLIP. Finally, we noted that SMAD1/5 were activated upon ALK5 inhibition in the presence of low levels of TGF-beta1, an effect associated with enhanced endothelial proliferation. We speculate that imbalance of ALK1 and ALK5 may contribute to the development of pulmonary artery hypertension.

Mechanism of C-type natriuretic peptide-induced endothelial cell hyperpolarization

C-type natriuretic peptide (CNP) has a demonstrated hyperpolarizing effect on vascular smooth muscle cells. However, its autocrine function, including its electrophysiological effect on endothelial cells, is not known. Here, we report the effect of CNP on the membrane potential (E(m)) of pulmonary microvascular endothelial cells and describe its target receptors, second messengers, and ion channels. We measured changes in E(m) using fluorescence imaging and perforated patch-clamping techniques. In imaging experiments, samples were preincubated in the potentiometric dye DiBAC(4)(3), and subsequently exposed to CNP in the presence of selective inhibitors of ion channels or second messengers. CNP exposure induced a dose-dependent decrease in fluorescence, indicating that CNP induces endothelial cell hyperpolarization. CNP-induced hyperpolarization was inhibited by the K(+) channel blockers, tetraethylammonium or iberiotoxin, the nonspecific cation channel blocker, La(3+), or by depletion or repletion of extracellular Ca(2+) or K(+), respectively. CNP-induced hyperpolarization was also blocked by pharmacological inhibition of PKG or by small interfering RNA (siRNA)-mediated knockdown of natriuretic peptide receptor-B (NPR-B). CNP-induced hyperpolarization was mimicked by the PKG agonist, 8-bromo-cGMP, and attenuated by both the endothelial nitric oxide synthase (eNOS) inhibitor, N(omega)-nitro-l-arginine methyl ester (l-NAME), and the soluble guanylyl cyclase (sGC) inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. Presence of iberiotoxin-sensitive, CNP-induced outward current was confirmed by perforated patch-clamping experiments. We conclude that CNP hyperpolarizes pulmonary microvascular endothelial cells by activating large-conductance calcium-activated potassium channels mediated by the activation of NPR-B, PKG, eNOS, and sGC.