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Troy Stevens

Publications and source records attributed to Troy Stevens.

At least 19 recordsLinked to original sources
PubMed2006

Activated leukocyte cell adhesion molecule is a component of the endothelial junction involved in transendothelial monocyte migration.

Transendothelial leukocyte migration is a major aspect of the innate immune response. It is essential in repair and regeneration of damaged tissues and is regulated by multiple cell adhesion molecules (CAMs) including members of the immunoglobulin (Ig) superfamily. Activated leukocyte cell adhesion molecule (ALCAM/CD166) is an Ig CAM expressed by activated monocytes and endothelial cells. Hitherto, the functional relevance of ALCAM expression by endothelial cells and activated monocytes remained unknown. In this report, we demonstrate soluble recombinant human ALCAM significantly inhibited the rate of transendothelial migration of monocyte cell lines. Direct involvement of ALCAM in transendothelial migration was evident from the robust inhibition of this process by ALCAM blocking antibodies. However, soluble recombinant ALCAM had no impact on monocyte migration or adhesion to endothelium. Localization of ALCAM specifically at cell-cell junctions in endothelial cells supported its role in transendothelial migration. This study is the first to localize ALCAM to endothelial cell junctions and demonstrate a functional relevance for co-expression of ALCAM by activated monocytes and endothelial cells.

Activated-Leukocyte Cell Adhesion Molecule↗
PubMed2006

Soluble adenylyl cyclase reveals the significance of cAMP compartmentation on pulmonary microvascular endothelial cell barrier.

Subtle elevations in cAMP localized to the plasma membrane intensely strengthen endothelial barrier function. Paradoxically, pathogenic bacteria insert adenylyl cyclases (ACs) into eukaryotic cells generating a time-dependent cytosolic cAMP-increase that disrupts rather than strengthens the endothelial barrier. These findings bring into question whether membrane versus cytosolic AC activity dominates in control of cell adhesion. To address this problem, a mammalian forskolin-sensitive soluble AC (sACI/II) was expressed in pulmonary microvascular endothelial cells. Forskolin stimulated this sACI/II construct generating a small cytosolic cAMP-pool that was not regulated by phosphodiesterases or Galphas. Whereas forskolin simultaneously activated the sACI/II construct and endogenous transmembrane ACs, the modest sACI/II activity overwhelmed the barrier protective effects of plasma membrane activity to induce endothelial gap formation. Retargeting sACI/II to the plasma membrane retained AC activity but protected the endothelial cell barrier. These findings demonstrate for the first time that the intracellular location of cAMP synthesis critically determines its physiological outcome.

Adenylyl Cyclases↗
PubMed2006

Regulation of endothelial cell barrier function by store-operated calcium entry.

Transient receptor potential (TRP) channels fulfill important and diverse signaling functions, and are generally conserved among species. The canonical subfamily of TRP proteins, TRPC channels, possesses 7 isoforms that combine in various ways to form heteromultimers. In endothelium, TRPC1 and TRPC4 form subunits of a channel that selectively conducts calcium. This channel is activated by calcium depletion in the endoplasmic reticulum, and thus TRPC1/TRPC4 forms the molecular basis of a store operated calcium entry pathway. TRPC4 interacts with protein 4.1, which tethers the channel to the membrane skeleton and represents a gating mechanism required for calcium permeation. In response to inflammatory agonists such as thrombin and bradykinin, the generation of inositol 1,4,5-trisphosphate transiently depletes endoplasmic reticulum calcium and activates the TRPC1/TRPC4 channel. Calcium permeation through this channel triggers cytoskeletal reorganization that is necessary to disrupt the endothelial cell barrier and increase permeability. Thus, inhibition of the TRPC1/TRPC4 channel provides a putative anti-inflammatory strategy.

Animals↗
PubMed2006

Hydraulic conductance of pulmonary microvascular and macrovascular endothelial cell monolayers.

Endothelial cells isolated from pulmonary arteries (RPAEC) and microcirculation (RPMVEC) of rat lungs were grown to confluence on porous filters and mounted on an Ussing-type chamber. Transmembrane pressure (deltaP) was controlled by the reservoir height, and the filtration rate corrected for surface area (J(v)/A) was measured by timing fluid movement in a calibrated micropipette. These parameters were used to calculate hydraulic conductance (Lp) by using linear regression of J(v)/A on deltaP. Mean Lp values for newly confluent RPAEC monolayers were 22 times higher than those for RPMVEC monolayers (28.6 +/- 5.6 vs. 1.30 +/- 0.50 x 10(-7) cm x s(-1) x cmH2O(-1); P < or = 0.01). After confluence was reached, electrical resistance and Lp remained stable in RPAEC but continued to change in RPMVEC with days in culture. Both phenotypes exhibited an initial time-dependent sealing response, but Lp also had an inverse relationship to deltaP in RPMVEC monolayers > or = 4 days postconfluence that was attributed to cell overgrowth rather than junctional length. In a comparison of the cadherin contents, E-cadherin was predominant in RPMVEC, but VE-cadherin was predominant in RPAEC. At a constant deltaP of 40-45 cmH2O for 2 h, J(v)/A increased 225% in RPAEC monolayers but did not change significantly in RPMVEC monolayers. Significant decreases in Lp were obtained after treatment with 5% albumin, GdCl3, or isoproterenol plus rolipram in both phenotypes. Thus lung microvascular endothelial cells exhibited a significantly lower Lp than conduit vessel endothelium, which would limit alveolar flooding relative to perivascular edema cuff formation during increased pulmonary vascular pressures.

Adrenergic beta-Agonists↗
PubMed2006

Cell-surface protein disulfide isomerase is required for transnitrosation of metallothionein by S-nitroso-albumin in intact rat pulmonary vascular endothelial cells.

S-nitrosation of the metal binding protein, metallothionein (MT) appears to be a critical link in affecting endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS)-derived nitric oxide (NO)-induced changes in cytoplasmic and nuclear labile zinc, respectively. Although low molecular weight S-nitrosothiols also appear to affect this signaling system, less is known about the ability of extracellular protein nitrosothiols to transnitrosate MT. Accordingly, we synthesized fluorescently labeled S-nitroso-albumin (SNO-albumin, a major protein S-nitrosothiol in plasma) and determined, via confocal microscopy in fixed tissue, that it is transported into cultured rat pulmonary vascular endothelial cells in a temperature sensitive fashion. The cells were transfected with an expression vector that encodes human MT-IIa cDNA sandwiched between enhanced cyan (donor) and yellow (acceptor) fluorescent proteins (FRET-MT) that can detect conformational changes in MT through fluorescence resonance energy transfer (FRET). SNO-albumin and the membrane-permeant low molecular weight S-nitroso-l-cysteine ethyl ester (l-SNCEE) caused a conformational change in FRET-MT as ascertained by full spectral laser scanning confocal microscopy in live rat pulmonary vascular endothelial cells, a result which is consistent with transnitrosation of the reporter molecule. Transnitrosation of FRET-MT by SNO-albumin, but not l-SNCEE, was sensitive to antisense oligonucleotide-mediated inhibition of the expression of cell surface protein disulfide isomerase (csPDI). These results extend the original observations of Ramachandran et al. (Ramachandran N, Root P, Jiang XM, Hogg PJ, Mutus B. Proc Natl Acad Sci U S A 98: 9539-9544, 2001) and suggest that csPDI-mediated denitrosation helps to regulate the ability of the major plasma NO carrier (SNO-albumin) to transnitrosate endothelial cell molecular targets (e.g. MT).

Animals↗
PubMed2005

Downregulation of endothelin-1 by farnesoid X receptor in vascular endothelial cells.

The farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily that is highly expressed in liver, kidney, adrenals, and intestine. FXR may play an important role in the pathogenesis of cardiovascular diseases via regulating the metabolism and transport of cholesterol. In this study, we report that FXR is also expressed in rat pulmonary artery endothelial cells (EC), a "nonclassical" bile acid target tissue. FXR is functional in EC, as demonstrated by induction of its target genes such as small heterodimer partner (SHP) after treatment with chenodeoxycholic acid, a FXR agonist. Interestingly, activation of FXR in EC led to downregulation of endothelin (ET)-1 expression. Reporter assays showed that activation of FXR inhibited transcriptional activation of the human ET-1 gene promoter and also repressed the activity of a heterologous promoter driven by activator protein (AP)-1 response elements. Electrophoretic mobility-shift and chromatin immunoprecipitation assays indicated that FXR reduced the binding activity of AP-1 transcriptional factors, suggesting that FXR may suppress ET-1 expression via negatively interfering with AP-1 signaling. These studies suggest that FXR may play a role in endothelial homeostasis and may serve as a novel molecular target for manipulating ET-1 expression in vascular EC.

Animals↗
PubMed2005

Activation of the endothelial store-operated ISOC Ca2+ channel requires interaction of protein 4.1 with TRPC4.

Store-operated calcium (SOC) entry represents the principal Ca2+ entry pathway into nonexcitable cells. Despite intensive investigation, mechanisms underlying activation of SOC entry have remained elusive. The endothelial ISOC channel is a Ca2+-selective SOC entry channel to which the transient receptor potential (TRP) proteins TRPC1 and TRPC4 contribute subunits. Activation of ISOC is specifically regulated by the spectrin-actin membrane skeleton; however, the nature of coupling between the ISOC channel and membrane skeleton is unknown. Here we demonstrate that protein 4.1 is an essential component of the ISOC channel gating mechanism. Protein 4.1 interacts with TRPC4 and the membrane skeleton. Deletion of the protein 4.1 binding domain on TRPC4 or peptide competition to the protein 4.1 binding domain prevents ISOC activation. These findings reveal that interaction of protein 4.1 with TRPC4 is required for activation of the endothelial ISOC channel.

Binding Sites↗
PubMed2005

Cyclic GMP-specific phosphodiesterase 5 regulates growth and apoptosis in pulmonary endothelial cells.

Sustained increases in intracellular cGMP concentrations ([cGMP]i) inhibit cell growth and induce apoptosis. We now report that a cGMP-specific phosphodiesterase, PDE5, plays a dominant role in regulating [cGMP]i transitions that inhibit cell growth and control susceptibility to apoptosis in pulmonary endothelium. Atrial natriuretic peptide (ANP) activates guanylyl cyclase A/B and induces a rapid [cGMP]i rise 2-5 min after its application, in both pulmonary arterial endothelial cells (PAECs) and pulmonary microvascular endothelial cells (PMVECs). However, increased [cGMP]i in PAECs is transient and decays within 10 min due to cytosolic PDE5 hydrolytic activity. Increased [cGMP]i in PMVECs is sustained for >3 h due to the absence of PDE5. Indeed, at any ANP concentration, the sustained (30 min) [cGMP]i rise is greater in PMVECs than in PAECs, unless PAECs are also treated with the PDE5 inhibitor zaprinast. Using RT-PCR, Western blot analysis, immunoprecipitation, and DEAE chromatography, we resolved the expression and activity of PDE 5A1/A2 only in PAECs. Similarly, PDE5 expression was restricted to extra-alveolar endothelium in vivo. ANP induced growth inhibition and apoptosis in PMVECs, but similar effects were not seen in PAECs unless ANP treatment was combined with zaprinast. ANP blocked the VEGF-induced proliferation and migration in PMVECs. Collectively, these data suggest that PDE5-regulated [cGMP]i controls endothelial cell growth and apoptosis, representing a mechanism of heterogeneity between two endothelial phenotypes.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2005

Essential role of a Ca2+-selective, store-operated current (ISOC) in endothelial cell permeability: determinants of the vascular leak site.

Store-operated calcium (SOC) entry is sufficient to disrupt the extra-alveolar, but not the alveolar, endothelial cell barrier. Mechanism(s) underlying such insensitivity to transitions in cytosolic calcium ([Ca2+]i) in microvascular endothelial cells are unknown. Depletion of stored Ca2+ activates a larger SOC entry response in extra-alveolar (pulmonary artery; PAECs) than alveolar (pulmonary microvascular; PMVECs) endothelial cells. In vivo permeation studies revealed that Ca2+ store depletion activates similar nonselective cationic conductances in PAECs and PMVECs, while only PAECs possess the calcium-selective, store-operated Ca2+ entry current, I(SOC). Pretreatment with the type 4 phosphodiesterase inhibitor, rolipram, abolished thapsigargin-activated I(SOC) in PAECs, and revealed I(SOC) in PMVECs. Rolipram pretreatment shifted the thapsigargin-induced fluid leak site from extra-alveolar to alveolar vessels in the intact pulmonary circulation. Thus, our results indicate I(SOC) provides a [Ca2+]i source that is needed to disrupt the endothelial cell barrier, and demonstrate that intracellular events controlling I(SOC) activation coordinate the site-specific vascular response to inflammation.

Adenylyl Cyclases↗
PubMed2005

Molecular and cellular determinants of lung endothelial cell heterogeneity.

Idiopathic pulmonary arterial hypertension is a progressive and potentially fatal disease with a limited number of therapeutic options. Two key lesions underlie the pathophysiology of this disease. The principal lesion is found in large- and intermediate-sized blood vessels and is characterized by medial and adventitial hypertrophy/hyperplasia, with distal extension of smooth-muscle layers into normally nonmuscularized vessels. The second lesion, found prominently in severe forms of pulmonary hypertension, originates in small precapillary vessel segments, commonly at blood vessel bifurcations. This "plexiform lesion" is a lumen-obliterative lesion comprised, at least in part, of cells that share endothelial cell attributes, but that have lost the "law of the monolayer." Indeed, the endothelial contribution to the (mal-)adaptive response in pulmonary hypertension is becoming increasingly apparent, with evidence that endothelium plays an important role in promoting the vasoconstriction and hyperproliferation of medial and adventitial cell layers in large- and intermediate-vessel sizes, and lumen obliteration in the plexiform lesion. Recent evidence indicates endothelial cells along the pulmonary artery and precapillary segments are phenotypically distinct and may fulfill different roles in these site-specific lesions. Thus, the present review summarizes our current understanding of pulmonary endothelial cell heterogeneity and discusses the potential role(s) of endothelial cell heterogeneity in the pathogenesis of pulmonary hypertension.

Endothelium, Vascular↗
PubMed2004

Stat3 activity is required for centrosome duplication in chinese hamster ovary cells.

The centrosome is the major microtubule organizing center in mammalian cells. During interphase, the single centrosome is duplicated and the progeny centrosomes then serve as the spindle poles during mitosis. Little is known about the signals that drive centrosome doubling. In these studies, various inhibitors and molecular approaches were used to demonstrate a role for the Stat pathway in regulating the events of centrosome doubling. Both piceatannol and a dominant negative behaving Stat3 adenovirus were able to disrupt centrosome duplication in hydroxyurea-arrested Chinese hamster ovary cells, demonstrating that Stat3 is a key signaling molecule in the events of centrosome duplication. Investigation into the role of Stat3 signaling during centrosome production demonstrated that Stat3 does not directly regulate the transcription of the centrosome genes encoding gamma-tubulin and PCM-1. Instead, Stat3 apparently regulated gamma-tubulin levels through post-transcriptional mechanisms whereas PCM-1 levels actually increased when Stat3 was inhibited, suggesting more complex mechanisms for regulating PCM-1 production. These studies demonstrate that Stat3 plays a vital role in centrosome duplication events, although the downstream targets of Stat3 activation leading to centrosome production remain to be established. The proposed signaling pathway utilizes Stat3 as a fundamental signaling molecule that directs the production of the various centrosome proteins indirectly.

Animals↗
PubMed2004

Activated leukocyte cell adhesion molecule in breast cancer: prognostic indicator.

INTRODUCTION: Activated leukocyte cell adhesion molecule (ALCAM) (CD166) is an immunoglobulin molecule that has been implicated in cell migration. The present study examined the expression of ALCAM in human breast cancer and assessed its prognostic value. METHODS: The immunohistochemical distribution and location of ALCAM was assessed in normal breast tissue and carcinoma. The levels of ALCAM transcripts in frozen tissue (normal breast, n = 32; breast cancer, n = 120) were determined using real-time quantitative PCR. The results were then analyzed in relation to clinical data including the tumor type, the grade, the nodal involvement, distant metastases, the tumor, node, metastasis (TNM) stage, the Nottingham Prognostic Index (NPI), and survival over a 6-year follow-up period. RESULTS: Immunohistochemical staining on tissue sections in ducts/acini in normal breast and in breast carcinoma was ALCAM-positive. Differences in the number of ALCAM transcripts were found in different types of breast cancer. The level of ALCAM transcripts was lower (P = 0.05) in tumors from patients who had metastases to regional lymph nodes compared with those patients without, in higher grade tumors compared with Grade 1 tumors (P < 0.01), and in TNM Stage 3 tumors compared with TNM Stage 1 tumors (P < 0.01). Tumors from patients with poor prognosis (with NPI > 5.4) had significantly lower levels (P = 0.014) of ALCAM transcripts compared with patients with good prognosis (with NPI < 3.4), and tumors from patients with local recurrence had significantly lower levels than those patients without local recurrence or metastases (P = 0.04). Notably, tumors from patients who died of breast cancer had significantly lower levels of ALCAM transcripts (P = 0.0041) than those with primary tumors but no metastatic disease or local recurrence. Patients with low levels of ALCAM transcripts had significantly (P = 0.009) more incidents (metastasis, recurrence, death) compared with patients with primary breast tumors with high levels of ALCAM transcripts. CONCLUSIONS: In the present panel of breast cancer specimens, decreased levels of ALCAM correlated with the nodal involvement, the grade, the TNM stage, the NPI, and the clinical outcome (local recurrence and death). The data suggest that decreased ALCAM expression is of clinical significance in breast cancer, and that reduced expression indicates a more aggressive phenotype and poor prognosis.

Activated-Leukocyte Cell Adhesion Molecule↗
PubMed2004

Paradoxical cAMP-induced lung endothelial hyperpermeability revealed by Pseudomonas aeruginosa ExoY.

Mammalian transmembrane adenylyl cyclases synthesize a restricted plasmalemmal cAMP pool that is intensely endothelial barrier protective. Bacteria have devised mechanisms of transferring eukaryotic factor-dependent adenylyl cyclases into mammalian cells. Pseudomonas aeruginosa ExoY is one such enzyme that catalyzes cytosolic cAMP synthesis, with unknown function. Pseudomonas aeruginosa genetically modified to introduce only the ExoY toxin elevated cAMP 800-fold in pulmonary microvascular endothelial cells over 4 hours, whereas a catalytically deficient (ExoY(K81M)) strain did not increase cAMP. ExoY-derived cAMP was localized to a cytosolic microdomain not regulated by phosphodiesterase activity. In contrast to the barrier-enhancing actions of plasmalemmal cAMP, the ExoY cytosolic cAMP pool induced endothelial gap formation and increased the filtration coefficient in the isolated perfused lung. These findings collectively illustrate a previously unrecognized mechanism of hyperpermeability induced by rises in cytosolic cAMP.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2004

CpG DNA-mediated immune response in pulmonary endothelial cells.

Although the CpG DNA immune response mediated by Toll-like receptor 9 (TLR9) has been extensively studied in a number of immune cells, the response to CpG DNA in endothelial cells (EC) is not well understood. In this study, we show that both mouse and rat lung EC display constitutive expression of TLR9 mRNA. Exposure to CpG DNA induced a potent proinflammatory response as manifested by an increased expression of IL-8 and ICAM-1 in mouse pulmonary EC. The proinflammatory response was sensitive to chloroquine, consistent with a role of endosomal contribution. A role for p38 MAPK and NF-kappaB pathway was apparent as the response was sensitive to inhibitors of p38 MAPK and NF-kappaB but was not affected by inhibitors of ERK1/2. A synergistic effect of CpG DNA and LPS on the inflammatory response is consistent with multiple TLR interaction in EC. This study suggests a possible role for CpG DNA-mediated EC immune response in the host defense system. It also has important implications in plasmid DNA-mediated pulmonary endothelium gene transfer.

Animals↗
PubMed2004

Paired-related homeobox gene Prx1 is required for pulmonary vascular development.

Herein, we show that the paired-related homeobox gene, Prx1, is required for lung vascularization. Initial studies revealed that Prx1 localizes to differentiating endothelial cells (ECs) within the fetal lung mesenchyme, and later within ECs forming vascular networks. To begin to determine whether Prx1 promotes EC differentiation, fetal lung mesodermal cells were transfected with full-length Prx1 cDNA, resulting in their morphological transformation to an endothelial-like phenotype. In addition, Prx1-transformed cells acquired the ability to form vascular networks on Matrigel. Thus, Prx1 might function by promoting pulmonary EC differentiation within the fetal lung mesoderm, as well as their subsequent incorporation into vascular networks. To understand how Prx1 participates in network formation, we focused on tenascin-C (TN-C), an extracellular matrix (ECM) protein induced by Prx1. Immunocytochemistry/histochemistry showed that a TN-C-rich ECM surrounds Prx1-positive pulmonary vascular networks both in vivo and in tissue culture. Furthermore, antibody-blocking studies showed that TN-C is required for Prx1-dependent vascular network formation on Matrigel. Finally, to determine whether these results were relevant in vivo, we examined newborn Prx1-wild-type (+/+) and Prx1-null (-/-) mice and showed that Prx1 is critical for expression of TN-C and lung vascularization. These studies provide a framework to understand how Prx1 controls EC differentiation and their subsequent incorporation into functional pulmonary vascular networks.

Animals↗