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Biomedical subjects

D English

Publications and source records attributed to D English.

At least 19 recordsLinked to original sources
PubMed2001

Platelet-released phospholipids link haemostasis and angiogenesis.

Considerable attention has focused on identifying mediators of neovascularization at sites of growth and abnormal tissue development. By contrast, mediators of angiogenesis at sites of injury and wound repair are not well defined but factors generated during blood coagulation (haemostasis) are attractive candidates. In addition to proteins generated, activated and released during the activation of clotting cascades, platelet-derived lipid mediators are now known to play a key role in many aspects of the angiogenic response. The first indication of lipid mediator involvement in angiogenesis was the discovery that lysophosphatidate (LPA), phosphatidic acid (PA) and sphingosine 1-phosphate (SPP) are high affinity agonists for G-protein coupled EDG (endothelial differentiation gene) receptors. The prototype for this family, EDG-1, was cloned from genes expressed when endothelial cells were activated to assume an angiogenic phenotype in vitro. The subsequent finding that SPP is a high affinity ligand for EDG-1 led Spiegel, Hla and associates (Lee et al., Science 1998;279:1552-1555) to hypothesize that platelet-released phospholipids play an important role in angiogenesis. These investigators and others demonstrated that SPP, LPA and phosphatidate (PA) induce many important endothelial cell responses associated with angiogenesis, including liberation of endothelial cells from established monolayers, chemotactic migration, proliferation, adherens junction assembly and morphogenesis into capillary-like structures. Although these studies indicated the potential involvement of platelet-derived phospholipids in angiogenesis, their physiological importance was not established. However, recent work demonstrates that >80% of the potent endothelial cell chemoattractive activity generated in human serum during clotting--an activity necessary for optimal angiogenesis--results from platelet-derived SPP. Other factors released from platelets during clotting, including LPA and PA, exert profound effects on endothelial cells that contribute unique aspects to the angiogenic response. These combined studies establish that SPP and other platelet-derived lipid mediators provide a novel link between haemostasis and angiogenesis.

Apoptosis↗
PubMed2001

Effect of phosphatidic acid on human breast cancer cells exposed to doxorubicin.

We previously demonstrated that phosphatidic acid (PA) induces chemotactic migration of highly metastatic breast cancer cells MDA-MB-231. The widely used anticancer drug doxorubicin was reported to induce apoptosis of cancer cells. Growth factors such as epidermal growth factor (EGF) and bioactive lipids such as lysophosphatidic acid (LPA) and sphingosine 1-phosphate (SPP) have been shown to enhance viability and to protect cancer cells against apoptosis. In this study, we investigated the effect of PA on MDA-MB-231 cells exposed to the anticancer drug doxorubicin. Cell migration toward PA was partially inhibited by doxorubicin treatment, and PA moderately diminished cell cycle arrest of cells exposed to doxorubicin. Although PA itself was not able to induce apoptosis of MDA-MB-231 cells, apoptosis of cells exposed to doxorubicin was markedly enhanced by PA treatment. Thus, PA is able to increase the apoptotic potential of doxorubicin, and may regulate the effects of doxorubicin used for chemotherapy.

Antibiotics, Antineoplastic↗
PubMed2001

The effect of repeated exposure to particulate air pollution (PM10) on the bone marrow.

Studies have shown that exposure to ambient particulate matter is related to an increased cardiopulmonary morbidity and mortality. The present study was designed to measure the effect of repeated exposure to urban air particles (PM10) on the rate of production and release of polymorphonuclear leukocytes (PMN) from the bone marrow into the peripheral blood. Rabbits exposed to PM10 (5 mg) twice a week for 3 wk, were given a bolus of 5'-bromo-2'-deoxyuridine (BrdU) to label dividing cells in the marrow that allows us to calculate the transit time of PMN in the bone marrow mitotic and postmitotic pools. The PM10 exposure (n = 8) causes a persistent increase in circulating band cells (p < 0.05) and a shortening of the transit time of PMN through the postmitotic pool in the marrow (64.4 +/- 2.2 h to 56.3 +/- 2.2 h, p < 0.05) if compared with vehicle-exposed control subjects (n = 6). PM10 exposure increases the bone marrow pool of PMN particularly the mitotic pool of PMN (p < 0.05). The PM10 were distributed diffusely in the lung and caused a mild mononuclear inflammation. The percentage of alveolar macrophages containing PM10 correlated significantly with the bone marrow PMN pool size (total pool r2 = 0.56, p < 0.012, mitotic pool r2 = 0.61, p < 0.007) and the transit time of PMN through the postmitotic pool (r2 = -0.42, p < 0.043). We conclude that repeated exposure to PM10 stimulates the bone marrow to increase the production of PMN in the marrow and accelerate the release of more immature PMN into the circulation. The magnitude of these changes was related to the amount of particles phagocytosed by alveolar macrophages.

Air Pollution↗
PubMed2001

Sphingosine 1-phosphate promotes endothelial cell barrier integrity by Edg-dependent cytoskeletal rearrangement.

Substances released by platelets during blood clotting are essential participants in events that link hemostasis and angiogenesis and ensure adequate wound healing and tissue injury repair. We assessed the participation of sphingosine 1-phosphate (Sph-1-P), a biologically active phosphorylated lipid growth factor released from activated platelets, in the regulation of endothelial monolayer barrier integrity, which is key to both angiogenesis and vascular homeostasis. Sph-1-P produced rapid, sustained, and dose-dependent increases in transmonolayer electrical resistance (TER) across both human and bovine pulmonary artery and lung microvascular endothelial cells. This substance also reversed barrier dysfunction elicited by the edemagenic agent thrombin. Sph-1-P-mediated barrier enhancement was dependent upon G(ialpha)-receptor coupling to specific members of the endothelial differentiation gene (Edg) family of receptors (Edg-1 and Edg-3), Rho kinase and tyrosine kinase-dependent activation, and actin filament rearrangement. Sph-1-P-enhanced TER occurred in conjunction with Rac GTPase- and p21-associated kinase-dependent endothelial cortical actin assembly with recruitment of the actin filament regulatory protein, cofilin. Platelet-released Sph-1-P, linked to Rac- and Rho-dependent cytoskeletal rearrangement, may act late in angiogenesis to stabilize newly formed vessels, which often display abnormally increased vascular permeability.

Actin Depolymerizing Factors↗
PubMed2001

A handbook for student nurses to guide clinical experiences in the school setting.

For more than 30 years, nursing students have had the opportunity to have clinical experiences related to their course requirements in the Dallas Public Schools. The Dallas Independent School District School Health Services Department staff provide an orientation to student nurses before their first day in the school clinic. To enhance their learning experience and clarify the regulations and expectations for student nurses, a handbook was prepared for the use of school nurses and the students. The Basic Health Care for the School-age Child: A Handbook for Student Nurses outlines the use of the school as a clinical experience setting. Another purpose for the handbook is to reduce the stress of this clinical rotation for the student nurse and for the staff nurse who serves as the student nurse's preceptor. This article describes the development of the expectations for the clinical experience and the information included in the handbook. An outline of the material included in each section is presented to provide ideas for school nurses who provide or are considering providing a rotation for student nurses in their schools.

Communicable Disease Control↗
PubMed2000

Sphingosylphosphorylcholine induces endothelial cell migration and morphogenesis.

Sphingosylphosphorylcholine (SPC) is one of the biologically active phospholipids that may act as extracellular messengers. Particularly important is the role of these lipids in the angiogenic response, a complex process involving endothelial cell migration, proliferation, and morphologic differentiation. Here we demonstrate that SPC and its hydrolytic product, sphingosine, induce chemotactic migration of human and bovine endothelial cells. The response is approximately equal to that elicited by vascular endothelial cell growth factor. The effect of SPC and sphingosine was associated with a rapid down-regulation of Edg1, a sphingosine 1-phosphate (SPP)-specific receptor involved in endothelial cell chemotaxis. Both SPC and sphingosine induced differentiation of endothelial cells into capillary-like structures in vitro. Thus, SPC and sphingosine join SPP among the biologically active lipids with angiogenic potential. Since neuronal abnormalities accompany pathological accumulation of SPC in brain tissue, it is possible that SPC is a modulator of angiogenesis in neural tissue upon its release from brain cells following trauma or neoplastic growth.

Animals↗
PubMed2000

Enhancement of the migration of metastatic human breast cancer cells by phosphatidic acid.

Phosphatidic acid (PA), lysophosphatidic acid (LPA), and sphingosine 1-phosphate (SPP) are naturally occurring phospholipids which induce a variety of effects as extracellular messengers. In this study, we compared the effects of these phospholipid signaling molecules on the migration of invasive and noninvasive breast cancer cell lines, an index of the metastatic potential of these cells. As previously demonstrated, invasive MDA-MB-231 breast cancer cells exhibited increased constitutive (nonstimulated) migration in comparison to poorly invasive MCF-7 cells. Phosphatidic acid employed at nanomolar concentrations markedly potentiated migration of the invasive cells but had no effect on migration of either the noninvasive MCF-7 cells or nonneoplastic human epithelial cells. Lysophosphatidic acid and sphingosine 1-phosphate inhibited both the directed (chemotactic) and random (chemokinetic) migration of MDA-MB-231 cells. Experiments were undertaken to characterize the signaling pathway involved in constitutive and PA-stimulated migration of MDA-MB-231 cells. The tyrosine kinase inhibitors staurosporine and genistein inhibited constitutive and PA-induced migration in a dose-dependent manner, consistent with a role for tyrosine phosphorylation in the migratory response. In addition, the phosphatidylinositol (PI) 3' kinase inhibitors wortmannin and LY294002 strongly inhibited both the constitutive and PA-stimulated migration of the invasive breast cancer cells, indicating that PI-3' kinase plays an important role in the metastatic migration of breast cancer cells. Finally, PA-induced migration of MDA-MB-231 was markedly attenuated by pretreatment of cells with Clostridium difficile Toxin B, pertussis toxin and suramin, implying a role for a Gi receptor-dependent process involving activation of the small GTP-binding protein Rho. Since an enhanced ability to migrate heightens the metastatic potential of cells within solid tumors, our results suggest that the metastatic capabilities of breast cancer cells may be enhanced by a receptor-driven cellular process initiated by phosphatidic acid or related lipid phosphate messengers.

Breast Neoplasms↗
PubMed2000

Phosphatidylinositol 3'-kinase-mediated calcium mobilization regulates chemotaxis in phosphatidic acid-stimulated human neutrophils.

Phosphatidylinositol 3'-kinase (PI 3'-kinase) plays an important role in the migration of hepatocytes, endothelial cells and neoplastic cells to agonists which activate cellular tyrosine kinases. We examined the PI 3'-kinase-dependent chemotactic responses of neutrophilic leukocytes induced by phosphatidic acid (PA) in order to clarify mechanisms by which the enzyme potentially influences cellular migration. Western analysis of immunoprecipitates indicated that PA induced the tyrosine phosphorylation of three distinct proteins involved in functional activation which co-immunoprecipitated in PA-stimulated cells. These proteins were identified as lyn, syk and the 85 kDa regulatory subunit of PI 3'-kinase. Chemotactic responses to PA but not to several other neutrophil agonists were inhibited by the PI 3'-kinase inhibitors wortmannin and LY294002. Chemotactic inhibition resulted from upstream inhibition of calcium mobilization. Chelation of extracellular calcium by ethylene glycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) did not affect the PA-induced chemotaxis, whereas chelation of intracellular calcium by 1, 2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) attenuated this response. Thus, changes in intracellular Ca(2+) levels that can be effected by Ca(2+) mobilized from intracellular stores in the absence of Ca(2+) influx regulate PA-induced chemotaxis. Furthermore, PI 3'-kinase inhibition blunted the agonist-dependent generation of inositol 1,4,5-trisphosphate (IP(3)), suggesting that PI 3'-kinase exerted its effects on calcium mobilization from intracellular sources by mediating activation of phospholipase C (PLC) in PA-stimulated cells. Moreover, the PI 3'-kinase inhibitor LY294002 also inhibited phosphorylation of syk in PA-stimulated cells. We, therefore, propose that products of PI 3'-kinase confined to the inner leaflet of the plasma membrane play a role in activation of syk, calcium mobilization and induction of chemotactic migration.

Calcium↗
PubMed2000

Polymorphonuclear leukocytes released from the bone marrow by granulocyte colony-stimulating factor: intravascular behavior.

INTRODUCTION: Granulocyte-colony stimulating factor (G-CSF) treatment stimulates the bone marrow and releases polymorphonuclear leukocytes (PMN) into the circulation. This study was designed to measure the intravascular margination, demargination and survival of PMN released from the marrow by G-CSF. MATERIALS AND METHODS: To trace PMN in the circulation, dividing PMN in the bone marrow of rabbits were labeled with 5'-bromo-2'-deoxyuridine (BrdU) and the effects of a single dose of G-CSF (12.5 microg/kg) on the behavior of these labeled cells in the circulation were measured. RESULTS: The results show that G-CSF induced a granulocytosis that peaked 12 h after treatment. This granulocytosis was associated with stimulation of the bone marrow characterized by shortening of the transit time of PMN through the marrow (97.3+/-2.5 h n=4 control vs 78.9+/-3.6 h n=5 G-CSF) particularly in the post-mitotic pool (P<0.01). Morphometric studies of the lung show a reduced sequestration of BrdU-labeled PMN in lung microvessels in G-CSF-treated animals (P<0.05) and a approximately 14-fold (G-CSF-group) vs a approximately 65-fold (control-group) enrichment of BrdU-labeled PMN in lung tissue if compared to circulating blood. The effect of G-CSF on demargination of PMN was measured by transferring BrdU-labeled PMN from donor animals treated with G-CSF to recipients. G-CSF did not cause demargination of intravascular PMN but delayed the clearance of G-CSF-treated PMN in the circulation. This delayed clearance was associated with inhibition of apoptosis in circulating PMN when measured both by morphology (17.7+/-2.3 vs 7.5+/-1.4%, P<0.01) and flow cytometry (16.2+/-1.1 vs 5+/-1.9%, P<0.01) using a DNA end-labeling method (control vs G-CSF group). CONCLUSION: We conclude that PMN released from the bone marrow by G-CSF sequestered less in the lung microvessels and have a prolonged intravascular life span.

Animals↗
PubMed2000

Sphingosine 1-phosphate released from platelets during clotting accounts for the potent endothelial cell chemotactic activity of blood serum and provides a novel link between hemostasis and angiogenesis.

Recent studies have identified factors responsible for angiogenesis within developing tumors, but mediators of vessel formation at sites of trauma, injury, and wound healing are not clearly established. Here we show that sphingosine 1-phosphate (S1P) released by platelets during blood clotting is a potent, specific, and selective endothelial cell chemoattractant that accounts for most of the strong endothelial cell chemotactic activity of blood serum, an activity that is markedly diminished in plasma. Preincubation of endothelial cells with pertussis toxin inhibited this effect of S1P, demonstrating the involvement of a Galphai-coupled receptor. After S1P-induced migration, endothelial cells proliferated avidly and differentiated forming multicellular structures suggestive of early blood vessel formation. S1P was strikingly effective in enhancing the ability of fibroblast growth factor to induce angiogenesis in the avascular mouse cornea. Our results show that blood coagulation initiates endothelial cell angiogenic responses through the release of S1P, a potent endothelial cell chemoattractant that exerts its effects by activating a receptor-dependent process.

Animals↗
PubMed2000

High-efficiency transient transfection of endothelial cells for functional analysis.

The definition of signaling pathways in endothelial cells has been hampered by the difficulty of transiently transfecting these cells with high efficiency. This investigation was undertaken to develop an efficient technique for the transfection of endothelial cells for functional analyses. Cells cotransfected with plasmid expressing green fluorescent protein (GFP) and the plasmid of interest were isolated by fluorescence-activated cell sorting (FACS) based on GFP expression. In the sorted cell population, a 2.5-fold enhancement in the number of cells expressing the gene of interest was observed, as confirmed by FACS analysis and Western blotting. Sorted cells retained functional properties, as demonstrated by chemotaxis to the agonist sphingosine 1-phosphate (SPP). To demonstrate the usefulness of this method for defining cellular signaling pathways, cells were cotransfected with plasmids encoding GFP and the carboxyl-terminal domain of the beta-adrenergic receptor kinase (beta ARKct), which inhibits signaling through the beta gamma dimer of heterotrimeric G-proteins. SPP-induced chemotaxis in sorted cells coexpressing beta ARKct was inhibited by 80%, demonstrating that chemotaxis was driven by a beta gamma-dependent pathway. However, no significant inhibition was observed in cells transfected with betaARKct but not enriched by sorting. Thus, we have developed a method for enriching transfected cells that allows the elucidation of crucial mechanisms of endothelial cell activation and function. This method should find wide applicability in studies designed to define pathways responsible for regulation of motility and other functions in these dynamic cells.

Animals↗