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Biomedical subjects

Edward F Plow

Publications and source records attributed to Edward F Plow.

At least 19 recordsLinked to original sources
PubMed2006

Genetic susceptibility to myocardial infarction and coronary artery disease.

Atherosclerotic involvement in the coronary arteries, which can result in heart attack and sudden death, is a common disease and prototypic of a complex human trait. To understand its genomic basis, eight linkage studies of sibling pairs have been performed. Although there was limited inter-study concordance of important loci, two gene variants in the leukotriene pathway (ALOX5AP and LTA4) have emerged as susceptibility factors for myocardial infarction (MI). Genome-wide association studies have also been undertaken, and the pro-inflammatory cytokine lymphotoxin-alpha (LTA), and its key ligand galectin-2 (LGALS2) have been identified as genes implicated in predisposition for heart attack. By cueing into the genomic basis for low serum LDL cholesterol levels, much work has been done to advance the importance of the serine protease PCSK9, which modulates LDL receptor function. Lifelong lowered LDL cholesterol associated with PCSK9 point mutations in 2-3% of individuals have been shown to provide marked protection from coronary artery disease (CAD). Most of the success in this field has been with the phenotype of MI, which is considerably more restrictive than CAD. Four principal and interdependent processes--lipoprotein handling, endothelial integrity, arterial inflammation, and thrombosis--have been supported as important via the clustering of genes, thus far implicated in CAD susceptibility. Of note, connecting genes in a single pathway (leukotriene), of a protein and its ligand (LTAalpha) or from one disease to another [age-related macular degeneration (AMD); complement factor H (CFH)], or even three disease characterized by inflammation (MHC2) have now been reported. Although the population attributable risk for any of the genes identified to date is limited, such discovery is likely to be accelerated in the future.

5-Lipoxygenase-Activating Proteins↗
PubMed2006

Identification of histone H2B as a regulated plasminogen receptor.

Tethering of plasminogen to cell surfaces controls plasmin formation and, thereby, influences pericellular proteolysis and cell migration. Modulation of cellular plasminogen binding sites provides a mechanism for regulation of these events. In this study, two distinct models, phorbol ester-stimulated adhesion of U937 monocytoid cells and culturing of peripheral blood neutrophils, treatments which modulate plasminogen binding sites, have been examined to determine the molecular basis for the upregulation of plasminogen receptors. Membranes were isolated from cell populations, with and without upregulated plasminogen binding capacities, and analyzed by [(125)I]plasminogen ligand blotting of gel transfers. Approximately 15 different [(125)I]plasminogen-binding proteins were discerned in the membrane fractions, and only relatively minor differences in the intensities of individual bands were noted in the different cell populations. The notable exception was the presence of a 17 kDa band, which was selectively and markedly enhanced in the membranes from cells with enhanced plasminogen binding capacities. The 17 kDa protein was isolated from both cell types, and amino acid sequencing of peptide fragments identified the same protein, histone H2B. Increased expression of histone H2B was observed on stimulated U937 cells and cultured neutrophils by confocal microscopy with an antibody raised to the carboxy-terminal octopeptide sequence of histone H2B. This antibody or its Fab fragments substantially decreased the level of binding of plasminogen to these cultured neutrophils and stimulated U937 cells that exhibited elevated levels of binding but not to nonstimulated cells. Thus, histone H2B represents a regulated plasminogen receptor, which contributes significantly to the plasminogen binding capacity of cells.

Amino Acid Sequence↗
PubMed2006

Regulation of integrin alphaIIbbeta3 activation by distinct regions of its cytoplasmic tails.

The short cytoplasmic tails regulate activation of integrin adhesion receptors via clasping/unclasping of their membrane-proximal helices. Using integrin alpha(IIb)beta(3) as a model, we show that a previously reported activating mutation alpha(IIb)(R(995)D) that perturbs the electrostatic interface in the clasp only partially activates alpha(IIb)beta(3) and that extensive activation of the receptor is achieved by complete deletion of alpha(IIb) CT or triple mutations in alpha(IIb)(V(990)A/F(992)A/R(995)D) that disrupt both electrostatic and hydrophobic interfaces in the clasp. The results provide quantitative evidence for an equilibrium-based integrin activation process where shifting the equilibrium to the fully activated state requires total unclasping of the cytoplasmic tails. We further demonstrate that while the C-terminal region of the alpha(IIb) tail minimally influences alpha(IIb)beta(3) activation, the C-terminal region of the beta(3) tail is critically involved. A disease-causing mutation of S(752)P in this region, but not S(752)A, suppressed partial activation induced by R(995)D or the talin head domain but did not affect activation induced by alpha(IIb) truncation. NMR spectroscopy revealed that S(752)P but not the S(752)A mutation disrupted a C-terminal helix within the beta(3) tail, suggesting that the C-terminal helix may regulate the equilibrium-based clasping/unclasping process. Together, these data provide molecular insights into how distinct regions of the cytoplasmic tails differentially and cooperatively regulate integrin activation.

Amino Acid Sequence↗
PubMed2006

The specificity and function of the metal-binding sites in the integrin beta3 A-domain.

The A-domains within integrin beta subunits contain three metal sites termed the metal ion-dependent adhesion site (MIDAS), site adjacent to the metal ion-dependent adhesion site (ADMIDAS), and ligand-induced metal-binding site (LIMBS), and these sites are involved in ligand engagement. The selectivity of these metal sites and their role in ligand binding have been investigated by expressing a fragment corresponding to the beta3 A-domain, beta3-(109-352), and single point mutants in which each of the cation-binding sites has been disabled. Equilibrium dialysis experiments identified three Mn2+- and two Ca2+-binding sites with the LIMBS being the site that did not bind Ca2+. Although the ADMIDAS could bind Ca2+, it did not bind Mg2+. These results indicate that the Ca2+-specific site that inhibits ligand binding is the ADMIDAS. Two different assay systems, surface plasmon resonance and a microtiter plate assay, demonstrated that the beta3 A-domain fragment bound fibrinogen in the presence of 0.1 mm Ca2+ but not in 3 mm Ca2+. This behavior recapitulated the effects of Ca2+ on fibrinogen binding to alphavbeta3 but not alphaIIbbeta3. Disabling any of the three cation-binding sites abrogated fibrinogen binding. These results indicate that the specificities of the three metal-binding sites for divalent cations are distinct and that each site can regulate the ligand binding potential of the beta3 A-domain.

Binding Sites↗
PubMed2006

Novel alpha1-adrenergic receptor signaling pathways: secreted factors and interactions with the extracellular matrix.

alpha1-Adrenergic receptor (alpha1-ARs) subtypes (alpha1A, alpha1B, and alpha1D) regulate multiple signal pathways, such as phospholipase C, protein kinase C (PKC), and mitogen-activated protein kinases. We employed oligonucleotide microarray technology to explore the effects of both short- (1 h) and long-term (18 h) activation of the alpha1A-AR to enable RNA changes to occur downstream of earlier well characterized signaling pathways, promoting novel couplings. Polymerase chain reaction (PCR) studies confirmed that PKC was a critical regulator of alpha1A-AR-mediated gene expression, and secreted interleukin (IL)-6 also contributed to gene expression alterations. We next focused on two novel signaling pathways that might be mediated through alpha1A-AR stimulation because of the clustering of gene expression changes for cell adhesion/motility (syndecan-4 and tenascin-C) and hyaluronan (HA) signaling. We confirmed that alpha1-ARs induced adhesion in three cell types to vitronectin, an interaction that was also integrin-, FGF7-, and PKC-dependent. alpha1-AR activation also inhibited cell migration, which was integrin- and PKC-independent but still required secretion of FGF7. alpha1-AR activation also increased the expression and deposition of HA, a glycosaminoglycan, which displayed two distinct structures: pericellular coats and long cable structures, as well as increasing expression of the HA receptor, CD44. Long cable structures of HA can bind leukocytes, which this suggests that alpha1-ARs may be involved in proinflammatory responses. Our results indicate alpha1-ARs induce the secretion of factors that interact with the extracellular matrix to regulate cell adhesion, motility and proinflammatory responses through novel signaling pathways.

Adrenergic alpha-1 Receptor Antagonists↗
PubMed2006

Cell adhesion and migration assays.

Adhesion and migration are basic responses of living cells to environmental stimuli. Such responses are central to a broad range of physiological processes, such as the immune response, repair of injured tissues, and prevention of excessive bleeding. Cell adhesion and migration also contributes to pathologies, including vascular and inflammatory diseases, as well as tumor growth and metastasis. These cellular responses depend on engagement of adhesion receptors by components of the extracellular matrix or molecules present on the surface of other cells. Hence, cell adhesion and migration assays are crucial methods in cell biology. In this chapter, several detailed protocols describing cell adhesion and migration assays are presented, and advantages and disadvantages of each method are discussed.

Cardiovascular Diseases↗
PubMed2005

Polymorphisms A387P in thrombospondin-4 and N700S in thrombospondin-1 perturb calcium binding sites.

Recent genetic studies have associated members of the thrombospondin (TSP) gene family with premature cardiovascular disease. The disease-associated polymorphisms lead to single amino acid changes in TSP-4 (A387P) and TSP-1 (N700S). These substitutions reside in adjacent domains of these highly homologous proteins. Secondary structural predictive programs and the homology of the domains harboring these amino acid substitutions to those in other proteins pointed to potential alterations of putative Ca2+ binding sites that reside in close proximity to the polymorphic amino acids. Since Ca2+ binding is critical for the structure and function of TSP family members, direct evidence for differences in Ca2+ binding by the polymorphic forms was sought. Using synthetic peptides and purified recombinant variant fragments bearing the amino acid substitutions, we measured differences in Tb3+ luminescence as an index of Ca2+ binding. The Tb3+ binding constants placed the TSP-1 region affected by N700S polymorphism among other high-affinity Ca2+ binding sites. The affinity of Ca2+ binding was lower for peptides (3.5-fold) and recombinant fragments (10-fold) containing the S700 vs. the N700 form. In TSP-4, the P387 form acquired an additional Ca2+ binding site absent in the A387 form. The results of our study suggest that both substitutions (A387P in TSP-4 and N700S in TSP-1) alter Ca2+ binding properties. Since these substitutions exert the opposite effects on Ca2+ binding, a decrease in TSP-1 and an increase in TSP-4, the two TSP variants are likely to influence cardiovascular functions in distinct but yet pathogenic ways.

Amino Acid Sequence↗
PubMed2005

Mechanism and effect of thrombospondin-4 polymorphisms on neutrophil function.

High-throughput genomic technology identified an association between a single nucleotide polymorphism (SNP), a proline (P387) rather than the predominant alanine (A387) at position 387 in thrombospondin-4 (TSP-4) and premature myocardial infarction. The inflammatory hypothesis of atherosclerosis invokes a prominent role of leukocytes and cytokines in pathogenesis. As the expression of TSP-4 by vascular cells permits its exposure to circulating leukocytes, the interactions of human neutrophils (polymorphonuclear leukocytes [PMNs]) with both TSP-4 variants were investigated. Phorbol 12-myristate 13-acetate (PMA)-stimulated PMNs adhered and migrated well and equally on the TSP-4 variants. Integrin alpha(M)beta2 was identified as the TSP-4 receptor mediating these responses, and the 3 epidermal growth factor (EGF)-like domains of TSP-4 harboring the SNPs interacted with the alpha(M)I-domain. Despite the similarity in these responses, the P387 variant induced more robust tyrosine phosphorylation of the stress-related mitogen-activated protein kinases (MAPKs): p38MAPK and c-Jun NH2-terminal kinase (JNK), as well as signal transducer and activator of transcription-1 (STAT1) and heat shock protein 27 (HSP27) than the A387 variant. Additionally, cells adherent to P387 TSP-4 variant released 4-fold more H2O2 and secreted 2-fold more interleukin 8 (IL-8) as compared with the A387. H2O2 release and p38MAPK activation were totally inhibited by blockade of alpha(M)beta2. Thus, alpha(M)beta2 plays a central role in proinflammatory activities of TSP-4 (P387) and may contribute to the prothrombotic phenotype associated with this variant.

Cell Adhesion↗
PubMed2005

Platelet, not endothelial, P-selectin is required for neointimal formation after vascular injury.

BACKGROUND: P-selectin blockade significantly inhibits inflammation and neointimal formation after arterial injury; however, the independent roles of platelet and endothelial P-selectins in this process are unknown. In atherosclerosis, both platelet and endothelial cell P-selectins are important. This study was designed to determine whether P-selectin expression on platelet, endothelial, or both surfaces is critical to the inflammatory response and neointimal formation after arterial injury. METHODS AND RESULTS: Using wild-type (WT) and P-selectin-knockout (Psel(-/-)) mice, we performed bone marrow transplantation to generate chimeric mice that expressed either platelet P-selectin (Plt-Psel) or endothelial P-selectin (EC-Psel). Double injury of the carotid artery was performed in these mice as well as in WT and Psel(-/-) mice. Animals were euthanized 4 or 21 days after arterial injury. Morphometric data showed that there was more neointimal formation in the WT mouse group when compared with the Psel(-/-) mouse group (0.015+/-0.004 vs 0.004+/-0.004 mm2, P<0.001). Further comparison showed significantly less neointimal area in EC-Psel mice (0.006+/-0.004 mm2) compared with Plt-Psel mice (0.011+/-0.005 mm2, P=0.026) and WT mice (0.015+/-0.004 mm2, P=0.001). No significant differences were observed between WT and Plt-Psel mice or between Psel(-/-) and EC-Psel mice. Decreased neointimal formation was accompanied by a reduced inflammatory response, as evidenced by immunostaining of RANTES and MCP-1 4 days after injury. CONCLUSIONS: Platelet P-selectin expression, but not endothelial P-selectin, plays a crucial role in the development of neointimal formation after arterial injury, and therapeutic strategies targeting leukocyte-platelet interactions could be effective in inhibiting restenosis.

Angioplasty, Balloon↗
PubMed2005

Thrombospondin-1 up-regulates expression of cell adhesion molecules and promotes monocyte binding to endothelium.

Expression of cell adhesion molecules (CAM) responsible for leukocyte-endothelium interactions plays a crucial role in inflammation and atherogenesis. Up-regulation of vascular CAM-1 (VCAM-1), intracellular CAM-1 (ICAM-1), and E-selectin expression promotes monocyte recruitment to sites of injury and is considered to be a critical step in atherosclerotic plaque development. Factors that trigger this initial response are not well understood. As platelet activation not only promotes thrombosis but also early stages of atherogenesis, we considered the role of thrombospondin-1 (TSP-1), a matricellular protein released in abundance from activated platelets and accumulated in sites of vascular injury, as a regulator of CAM expression. TSP-1 induced expression of VCAM-1 and ICAM-1 on endothelium of various origins, which in turn, resulted in a significant increase of monocyte attachment. This effect could be mimicked by a peptide derived from the C-terminal domain of TSP-1 and known to interact with CD47 on the cell surface. The essential role of CD47 in the cellular responses to TSP-1 was demonstrated further using inhibitory antibodies and knockdown of CD47 with small interfering RNA. Furthermore, we demonstrated that secretion of endogenous TSP-1 and its interaction with CD47 on the cell surface mediates endothelial response to the major proinflammatory agent, tumor necrosis factor alpha (TNF-alpha). Taken together, this study identifies a novel mechanism regulating CAM expression and subsequent monocyte binding to endothelium, which might influence the development of anti-atherosclerosis therapeutic strategies.

Atherosclerosis↗
PubMed2005

Identity of the amino acid residues involved in C3bi binding to the I-domain supports a mosaic model to explain the broad ligand repertoire of integrin alpha M beta 2.

Interactions between the complement degradation product C3bi and leukocyte integrin alpha(M)beta(2) are critical for host defense against foreign pathogens and in tumor cell surveillance. To gain insight into the mechanism by which the alpha(M)I-domain of the integrin interacts with C3bi, detailed mapping of the C3bi binding site was undertaken. Previous mutagenesis studies had implicated five small structural segments within the alpha(M)I-domain in recognition of this ligand. Sets of three amino acids within the five implicated segments were mutated to the corresponding alpha(L)I-domain residues. Then, within the affected mutants, single point mutations were introduced to precisely define the requisite residues. Ultimately, H148, F150, Q204, L205, R208, T211, T213, I256, P257 were identified as being critical for C3bi binding. A synthetic peptide approach confirmed the involvement of the specified residues with the complex midsegment, Q204-I215, in C3bi recognition. Furthermore, the alpha(D)I-domain, which has a low intrinsic affinity for C3bi, acquired high affinity for the ligand when the implicated residues were inserted. The residues necessary to engage C3bi reside on or adjacent to the cation binding MIDAS site of the alpha(M)I-domain. The amino acids involved in C3bi binding are distinct from those involved in interaction of previously mapped ligands with the alpha(M)I-domain. This divergence supports a mosaic model, in which different ligands engage different amino acids to bind to alpha(M)I-domain, accounting for the broad recognition capacity of integrin alpha(M)beta(2).

Amino Acid Sequence↗
PubMed2004

Transcription factor MEF2A mutations in patients with coronary artery disease.

Coronary artery disease (CAD), including its most serious complication myocardial infraction (MI), is the leading cause of death in the US and developed countries. We recently discovered that a seven-amino acid deletion in MEF2A, a transcription factor with a high level of expression in the endothelium of coronary arteries, co-segregates with CAD/MI in one family, and it suppresses transcription activation activity of MEF2A by a dominant-negative mechanism. In this study, we used single-strand conformation polymorphism and DNA sequence analyses to identify mutations in MEF2A in 207 independent CAD/MI patients and 191 controls with normal angiograms. We identified three novel mutations in exon 7 of MEF2A in four of 207 CAD/MI patients (1.93%). No mutations were detected in the 191 controls. The mutations identified here include N263S identified in two independent CAD patients, P279L in one patient and his father with the diagnosis of CAD and G283D in one patient. These mutations are clustered within or close to the major transcriptional activation domain of MEF2A. They significantly reduce the transcriptional activation activity of MEF2A and act by a loss-of-function mechanism. The gene carriers with loss-of-function mutations appear to be associated with less severe CAD. These results suggest that CAD/MI can result from a spectrum of MEF2A transcription dysfunctions ranging from loss-of-function to dominant-negative suppression and that a significant percent of the CAD/MI population (1.93%) may carry mutations in MEF2A, although further definition of the prevalence of MEF2A mutations is warranted.

Adult↗
PubMed2004

Distinct roles for the alpha and beta subunits in the functions of integrin alphaMbeta2.

Integrin alphaMbeta2 (Mac-1, CD11b/CD18) is a noncovalently linked heterodimer of alphaM and beta2 subunits on the surface of leukocytes, where it plays a pivotal role in the adhesion and migration of these cells. Using HEK293 cells expressing alphaMbeta2 or the individual constituent chains on their surface, we analyzed the contributions of the alphaM or beta2 subunits to functional responses mediated by the integrin. In cells expressing only alphaM or beta2, the individual subunits were not associated with the endogenous integrins of the cells, and other partners for the subunits were not detected by surface labeling and immunoprecipitation under a variety of conditions. The alphaM cells mediated adhesion and spreading on a series of alphaMbeta2 ligands (fibrinogen, Factor X, iC3b, ICAM-1 (intercellular adhesion molecule-1), and denatured ovalbumin) but could not support cell migration to any of these. The spreading of the alphaM cells suggested an unanticipated linkage of this subunit to the cytoskeleton. The beta2 cells supported migration and attachment but not spreading on a subset of the alphaMbeta2 ligands. The heterodimeric receptor and its individual subunits were purified from the cells by affinity chromatography and recapitulated the ligand binding properties of the corresponding cell lines. These data indicate that each subunit of alphaMbeta2 contributes distinct properties to alphaMbeta2 and that, in most but not all cases, the response of the integrin is a composite of the functions of its individual subunits.

CD11b Antigen↗
PubMed2004

P-selectin binding to P-selectin glycoprotein ligand-1 induces an intermediate state of alphaMbeta2 activation and acts cooperatively with extracellular stimuli to support maximal adhesion of human neutrophils.

P-selectin glycoprotein ligand 1 (PSGL-1, CD162) and integrin alphaMbeta2 (Mac-1, CD11bCD18) are leukocyte adhesion molecules essential for innate immunity and inflammation. The interaction of PSGL-1 with P-selectin (CD62P) mediates tethering, rolling, and weak adhesion of leukocytes, during which they become sufficiently activated in situ by locally released or displayed cytokines and chemoattractants for integrin-mediated firm adhesion. However, communication between P-selectin and the integrin, whether P-selectin can trigger beta2-integrin activation, remains controversial. We found that P-selectin immunoglobulin chimera and PSGL-1 monoclonal antibodies (mAbs) increased adhesion of human neutrophils to immobilized, but not soluble, fibrinogen. This intermediate state of neutrophil adhesion was defined by moderate clustering of integrin alphaMbeta2, no increase in CBRM1/5 (a mAb specific for the activation epitope on the alphaM subunit) recognition, and no increase in surface expression of alphaMbeta2, whereas phorbol myristate acetate (PMA) induced extensive changes in these 3 parameters. Furthermore, platelet-activating factor or interleukin 8 acted in concert with P-selectin for further enhancing the activation of alphaMbeta2. We thus propose a model in which P-selectin induces an intermediate state of integrin activation and then cooperates with other extracellular stimuli to support maximal adhesion of human neutrophils.

Antibodies↗
PubMed2004

Membrane-mediated structural transitions at the cytoplasmic face during integrin activation.

Cytoplasmic face-mediated integrin inside-out activation remains a paradigm in transmembrane signal transduction. Emerging evidence suggests that this process involves dissociation of the complex between the integrin cytoplasmic tails; however, a dynamic image of how it occurs on the membrane surface remains elusive. We show here that, whereas membrane-proximal helices of integrin alpha/beta cytoplasmic tails associate in cytoplasm-like aqueous medium, they become partially embedded into membrane-mimetic micelles when unclasped. Membrane embedding induces substantial structural changes of the cytoplasmic tails as compared to their aqueous conformations and suggests there may be an upward movement of the membrane-proximal helices into the membrane during their separation. We further demonstrate that the beta3 tail exhibits additional membrane binding site at its C terminus containing the NPLY motif. Talin, a key intracellular integrin activator, recognizes this site as well as the membrane-proximal helix, thereby promoting cytoplasmic tail separation along the membrane surface. These data provide a structural basis of membrane-mediated changes at the cytoplasmic face in regulating integrin activation and signaling.

Amino Acid Sequence↗
PubMed2004

Molecular and functional differences induced in thrombospondin-1 by the single nucleotide polymorphism associated with the risk of premature, familial myocardial infarction.

A serine (Ser-700) amino acid rather than an asparagine (Asn-700) at residue 700 of thrombospondin-1 has been linked to an increased risk for development of premature, familial heart attacks. We now have identified both functional and structural differences between the Ser-700 and Asn-700 thrombospondin-1 variants. The Ser-700 variant increased the rate and extent of platelet aggregation and showed increased surface expression on platelets compared with the Asn-700 variant. These differences could be ascribed to an enhanced interaction of the Ser-700 variant with fibrinogen on the platelet surface and are consistent with a prothrombotic phenotype in Ser-700 individuals. The Ser-700 variant thrombospondin-1 was conformationally more labile than the Asn-700 variant as demonstrated by increased susceptibility to proteolytic digestion and enhanced susceptibility to unfolding by denaturants. These data suggest a potential molecular and cellular basis for a genetic risk factor associated with early onset myocardial infarction.

Asparagine↗