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Lindsey A Miles

Publications and source records attributed to Lindsey A Miles.

13 recordsLinked to original sources
PubMed2006

Cell-surface actin binds plasminogen and modulates neurotransmitter release from catecholaminergic cells.

An emerging area of research has documented a novel role for the plasminogen activation system in the regulation of neurotransmitter release. Prohormones, secreted by cells within the sympathoadrenal system, are processed by plasmin to bioactive peptides that feed back to inhibit secretagogue-stimulated release. Catecholaminergic cells of the sympathoadrenal system are prototypic prohormone-secreting cells. Processing of prohormones by plasmin is enhanced in the presence of catecholaminergic cells, and the enhancement requires binding of plasmin(ogen) to cellular receptors. Consequently, modulation of the local cellular fibrinolytic system of catecholaminergic cells results in substantial changes in catecholamine release. However, mechanisms for enhancing prohormone processing and cell-surface molecules mediating the enhancement on catecholaminergic cells have not been investigated. Here we show that plasminogen activation was enhanced >6.5-fold on catecholaminergic cells. Carboxypeptidase B treatment decreased cell-dependent plasminogen activation by approximately 90%, suggesting that the binding of plasminogen to proteins exposing C-terminal lysines on the cell surface is required to promote plasminogen activation. We identified catecholaminergic plasminogen receptors required for enhancing plasminogen activation, using a novel strategy combining targeted specific proteolysis using carboxypeptidase B with a proteomics approach using two-dimensional gel electrophoresis, radioligand blotting, and tandem mass spectrometry. Two major plasminogen-binding proteins that exposed C-terminal lysines on the cell surface contained amino acid sequences corresponding to beta/gamma-actin. An anti-actin monoclonal antibody inhibited cell-dependent plasminogen activation and also enhanced nicotine-dependent catecholamine release. Our results suggest that cell-surface-expressed forms of actin bind plasminogen, thereby promoting plasminogen activation and increased prohormone processing leading to inhibition of neurotransmitter release.

Actins↗
PubMed2006

Plasminogen inhibits TNFalpha-induced apoptosis in monocytes.

Monocytes are major mediators of inflammation, and apoptosis provides a mechanism for regulating the inflammatory response by eliminating activated macrophages. Furthermore, as a consequence of apoptosis, plasminogen binding is markedly increased on monocytoid cells. Therefore, we investigated the ability of plasminogen to modulate monocyte apoptosis. Apoptosis of monocytoid cells (human monocytes and U937 cells) was induced with either TNFalpha or cycloheximide. When apoptosis was induced in the presence of increasing concentrations of plasminogen, apoptosis was inhibited in a dose-dependent manner with full inhibition achieved at 2 microM plasminogen. Plasminogen treatment also markedly reduced internucleosomal DNA fragmentation and reduced levels of active caspase 3, caspase 8, and caspase 9 induced by TNFalpha or by cycloheximide. We examined the requirement for plasmin proteolytic activity in the cytoprotective function of plasminogen. A plasminogen active site mutant, [D(646)E]-Plg, failed to recapitulate the cytoprotective effect of wild-type plasminogen. Furthermore, antibodies against PAR1 blocked the antiapoptotic effect of plasminogen. Our results suggest that plasminogen inhibits monocyte apoptosis. The cytoprotective effect of plasminogen requires plasmin proteolytic activity and requires PAR1. Because apoptosis of monocytes plays a key role in inflammation and atherosclerosis, these results provide insight into a novel role of plasminogen in these processes.

Amino Acid Substitution↗
PubMed2005

Plasminogen receptors: the sine qua non of cell surface plasminogen activation.

Localization of plasminogen and plasminogen activators on cell surfaces promotes plasminogen activation and serves to arm cells with the broad spectrum proteolytic activity of plasmin. Cell surface proteolysis by plasmin is an essential feature of physiological and pathological processes requiring extracellular matrix degradation for cell migration including macrophage recruitment during the inflammatory response, tissue remodeling, wound healing, tumor cell invasion and metastasis and skeletal myogenesis. Cell associated plasmin on platelets and endothelial cells is optimally localized for promotion of clot lysis. In more recently recognized functions that are likely to be independent of matrix degradation, cell surface-bound plasmin participates in prohormone processing as well as stimulation of intracellular signaling. This issue of Frontiers in Bioscience on Plasminogen Receptors encompasses chapters focusing on the kinetics of cell surface plasminogen activation and the regulation of plasminogen receptor activity as well as the contribution of plasminogen receptors to the physiological and pathophysiological processes of myogenesis, muscle regeneration and cancer. The molecular identity of plasminogen receptors is cell-type specific, with distinct molecular entities providing plasminogen receptor function on different cells. This issue includes chapters on the well studied plasminogen receptor functions.

Animals↗
PubMed2005

Reduction of myocardial infarct size by doxycycline: a role for plasmin inhibition.

Myocardial ischemia-reperfusion (I/R) is associated with the activation of matrix metalloproteinases (MMPs) and serine proteases. We hypothesized that activation of MMPs and the serine protease plasmin contribute to early cardiac myocyte death following I/R and that broad-spectrum protease inhibition with doxycycline (DOX) preserves myocyte viability. Rats treated daily with or without DOX beginning 48 h prior to experimentation were subjected to 30 min of coronary occlusion and 2 days of reperfusion. DOX pre-treatment reduced infarct size by 37%. DOX attenuated increases in MMP-9 and plasmin levels as determined by gelatin zymography and immunoblot, respectively. Neutrophil extravasation was unaltered by DOX as assessed by myeloperoxidase (MPO) activity. To examine the contribution of MMP-9 and plasmin to myocyte injury, cultures of neonatal rat ventricular myocytes (NRVMs) were treated for 48 h with 83 kDa MMP-9 or plasminogen in the presence or absence of DOX. MMP-9 treatment did not affect myocyte viability. Plasminogen treatment led to increased plasmin activity, resulting in loss of beta1-integrin, NRVM detachment and apoptosis. DOX co-treatment inhibited plasmin activity and preserved NRVM attachment, whereas co-treatment with the broad-spectrum MMP inhibitor GM6001 had no effect. These results indicate that plasmin causes disruption of myocyte attachment and viability independently of MMP activation in vitro and that inhibition of plasmin by DOX may reduce I/R-induced myocyte death in vivo through the inhibition of plasmin.

Animals↗
PubMed2003

Inhibition of cell surface mediated plasminogen activation by a monoclonal antibody against alpha-Enolase.

Localization of plasmin activity on leukocyte surfaces plays a critical role in fibrinolysis as well as in pathological and physiological processes in which cells must degrade the extracellular matrix in order to migrate. The binding of plasminogen to leukocytic cell lines induces a 30- to 80-fold increase in the rate of plasminogen activation by tissue-type (tPA) and urokinase-type (uPA) plasminogen activators. In the present study we have examined the role of alpha-enolase in plasminogen activation on the cell surface. We produced and characterized a monoclonal antibody (MAb) 11G1 against purified alpha-enolase, which abrogated about 90% of cell-dependent plasminogen activation by either uPA or tPA on leukocytoid cell lines of different lineages: B-lymphocytic, T-lymphocytic, granulocytic, and monocytic cells. In addition, MAb 11G1 also blocked enhancement of plasmin formation by peripheral blood neutrophils and monocytes. In contrast, MAb 11G1 did not affect plasmin generation in the presence of fibrin, indicating that this antibody did not interact with fibrinolytic components in the absence of cells. These data suggest that, although leukocytic cells display several molecules that bind plasminogen, alpha-enolase is responsible for the majority of the promotion of plasminogen activation on the surfaces of leukocytic cells.

Adenocarcinoma↗
PubMed2003

Critical role for conversion of glu-plasminogen to Lys-plasminogen for optimal stimulation of plasminogen activation on cell surfaces.

When Glu-plasminogen, the native circulating form of the zymogen, is bound to cell surfaces, its activation is markedly enhanced compared with the reaction in solution. This results in localization of the broad-spectrum proteolytic activity of plasmin on cell surfaces. The cell-associated plasmin plays a key role in fibrinolysis, cell migration, and prohormone processing. It is well established that the localization of plasminogen and plasminogen activators on cell surfaces promotes the enhanced plasminogen activation on the cell surface. The focus of this article is to review recent studies demonstrating that the conversion of Glu-plasminogen to the more readily activated Lys-plasminogen derivative is necessary for optimal stimulation of plasminogen activation on the cell surface, and that the interaction of Glu-plasminogen with cells serves to increase processing of Glu-plasminogen to Lys-plasminogen, thereby enhancing plasminogen activation on the cell surface.

Fibrinolytic Agents↗
PubMed2002

Localization of regulatory elements mediating constitutive and cytokine-stimulated plasminogen gene expression.

The activity of plasmin, the major enzyme responsible for dissolving fibrin clots, is regulated by plasminogen activators, plasminogen activator inhibitors, alpha(2)-antiplasmin, and inflammatory mediators. Recent studies suggest that plasmin activity can be regulated also at the level of plasminogen gene expression. In this study, we characterized the murine plasminogen promoter and 5'-flanking region. The major transcription start site was identified at -83 bp relative to the ATG translational initiation codon. A series of 5'-flanking sequences up to 2400 bp upstream of the transcription initiation site were fused to the luciferase reporter gene and transfected into hepatocytic cells. A 106-bp 5'-flanking region of the murine plasminogen gene demonstrated sufficient functional promoter activity in plasminogen-expressing cells. IL-6 treatment stimulated luciferase activity driven by the 5'-flanking region and an intact consensus IL-6-responsive element at -791, was required for maximal stimulation by this cytokine. These results indicate the presence of regulatory elements in the 5'-flanking region of the murine plasminogen promoter that may regulate murine plasminogen gene expression and, hence, plasmin activity.

Amino Acid Motifs↗
PubMed2002

Plasmin-induced migration of endothelial cells. A potential target for the anti-angiogenic action of angiostatin.

Angiostatin, a plasminogen fragment containing 3-4 N-terminal kringle domains, is a potent inhibitor of tumor-induced angiogenesis, but its mechanism of action is unclear. Angiostatin is a ligand for integrin alphavbeta(3) but does not induce stress fiber formation upon integrin binding, suggesting that angiostatin is a potential integrin antagonist. Plasmin, the parent molecule of angiostatin and a major extracellular protease, induces platelet aggregation, migration of peripheral blood monocytes, and release of arachidonate and leukotriene from several cell types. In the current study, we found that plasmin specifically bound to alphavbeta(3) through the kringle domains and induced migration of endothelial cells. In contrast, angiostatin did not induce cell migration. Notably, angiostatin, anti-alphavbeta(3) antibodies, RGD-peptide, and a serine protease inhibitor effectively blocked plasmin-induced cell migration. These results suggest that plasmin-induced migration of endothelial cells requires alphavbeta(3) and the catalytic activity of plasmin and that this process is a potential target for the inhibitory activity of angiostatin.

Angiogenesis Inhibitors↗
PubMed2002

The local chromaffin cell plasminogen/plasmin system and the regulation of catecholamine secretion.

Chromaffin cells express components of the plasminogen/plasmin system, including its major activator, tissue plasminogen activator (t-PA), and high-affinity cellular receptors for plasminogen, which promote local concentration and activation of plasminogen at the cell surface. Our studies suggest that plasmin participates in local neuroendocrine prohormone processing and that perturbation of this system profoundly affects the secretory characteristics of the cells. These results suggest the presence of a local, functionally active, chromaffin cell plasminogen/plasmin system that plays a major role in the regulation of catecholamine release from catecholaminergic cells.

Animals↗
PubMed2002

Plasminogen has a broad extrahepatic distribution.

Plasmin is the major enzyme that dissolves fibrin in the vasculature and the predominant source of its zymogen, plasminogen, is liver. However, plasmin has a broad substrate spectrum and, if present in other tissues, may perform additional functions. We tested the hypothesis that plasminogen is expressed broadly extrahepatically. A sensitive and specific isotopic quantitative RT-PCR assay was developed to detect plasminogen mRNA from total RNA isolated from C57BL/6J mice tissues. Plasminogen mRNA was detected in adrenal, kidney, brain, testis, heart, lung, uterus, spleen, thymus and gut. Of these tissues, adrenal had the highest plasminogen mRNA content. In situ hybridization was utilized to localize plasminogen mRNA expressing cell types. Besides hepatocytes, positive cells were identified in both adrenal and kidney medullae and cortexes. Plasminogen mRNA expression was detected in cerebral, hippocampal and cerebellar neurons. Plasminogen mRNA was detected in cells in early stages of spermatogenesis in testis, present in the cortex and medulla of the thymus and in splenic white and red pulps. Our results suggest that the plasminogen gene is expressed broadly in extrahepatic tissues. Thus, tissues separated by local anatomic barriers as well as tissues accessible to circulating plasminogen have the capacity to provide local sources of plasminogen.

Animals↗
PubMed2002

A single thymine nucleotide deletion responsible for congenital deficiency of plasmin inhibitor.

Plasma plasmin inhibitor (PI) is a physiological inhibitor of plasmin-mediated fibrinolysis and constitutes a hemostatic component in blood plasma; hence its deficiency results in a severe hemorrhagic diathesis. We have carried out molecular analysis of American family members with congenital PI deficiency, and detected a single thymine deletion at nucleotide position 332 in exon 5. The deletion was found in both alleles of the homozygotes and in one allele of the heterozygotes, and the patterns of restriction fragment length polymorphism created by the mutation in the family members were compatible with their phenotypes. The deletion caused a frameshift leading to an alteration and shortening of the deduced amino acid sequence. The amino acid sequence consists of the first 83 amino acids of the N-terminal sequence of the normal PI and additional new amino acids, resulting in a mutant composed of 94 amino acids in contrast to 464 amino acids of the normal PI. In transient expression analysis, the mutant PI whose molecular size was compatible with the predicted amino acid sequence was detected in the lysates of the cells transfected with the mutated PI expression vector. The mutant PI was retained and underwent progressive degradation within the cells, and was minimally excreted into the media. These data indicate that this mutation is the cause of PI deficiency in this pedigree.

Cloning, Molecular↗