Complement, oxidants, and endothelial injury: how a bedside observation opened a door to vascular biology.

A single encounter with a dialysis patient led to the study of complement and neutrophil aggregation, which in turn spawned our work and the remarkable development of the field of vascular biology. As our understanding of these cellular interactions and the signaling pathways involved in these processes has expanded, so has our appreciation for the broad impact of this work on an array of human diseases.

A single amino acid substitution changes ribonuclease 4 from a uridine-specific to a cytidine-specific enzyme.

The structural features underlying the strong uridine specificity of ribonuclease 4 (RNase 4) are largely unknown. It has been hypothesized that the negatively charged alpha-carboxylate is close to the pyrimidine binding pocket, due to a unique C-terminal deletion. This would suppress the cleavage of cytidine-containing substrates [Zhou, H.

Mechanism of tissue factor activation on HL-60 cells.

Tissue factor (TF) procoagulant activity (PCA) on the surface of intact HL-60 cells is encrypted. This latent TF PCA was activated by exposing the cells to ionomycin, a calcium ionophore. Within seconds an increase in TF PCA of greater than 100-fold was observed.

Apoptosis is associated with increased cell surface tissue factor procoagulant activity.

Tissue factor (TF), the physiologic initiator of coagulation, is present on the surface of cells but is not fully active unless the cell is lysed. This phenomenon, termed TF encryption, may be regulated by changes in membrane structure. Because apoptosis is associated with cell membrane alterations and conditions associated with apoptosis have also been associated with TF de-encryption, we hypothesized that apoptosis would result in enhanced TF procoagulant activity.

Training internal medicine residents in the community: the Minnesota experience.

The University of Minnesota Internal Medicine Residency Program has developed ambulatory general medicine rotations in rural community settings and urban managed care settings in Minnesota. Based on what had been learned from community focus groups, from discussions with residents about what they perceived to be training holes in the traditional curriculum, and from resident evaluations of pilot rotations, an educational framework for the rotations was established. The authors describe the process of developing these rotations, their educational rationale and objectives, the structure of the rotations, teaching strategies, faculty development, and the evaluation system, and outline the important elements of the successful implementation of these new rotations.

Soluble CD14 promotes LPS activation of CD14-deficient PNH monocytes and endothelial cells.

Bacterial lipopolysaccharide (LPS) initiates the cascade of inflammatory events that, in infected patients, often result in a lethal systemic inflammatory response known as the sepsis syndrome. We studied LPS-stimulated expression of tissue factor (TF) in human peripheral blood mononuclear cells (PBMCs) and cultured endothelial cells or tumor necrosis factor-alpha (TNF-alpha) in PBMCs. CD14, a PBMC membrane protein, is involved in LPS signaling and is also present as a soluble molecule in serum.

Induction of endothelial tissue factor by endotoxin and its precursors.

The structural determinants of lipopolysaccharide required for the induction of tissue factor in human umbilical vein endothelial cells were studied. Intact lipid A was essential for the induction of tissue factor whereas the incomplete lipid A precursors lipid IVA and lipid X, as well as monophosphoryl lipid A and acyloxyacyl hydrolase-treated lipopolysaccharide, were unable to induce tissue factor and tissue factor specific mRNA. However, the lipid A precursor, lipid IVA, was able to inhibit LPS-mediated induction of tissue factor; structural determinants distal to lipid A were found to be required for maximal induction of tissue factor activity and tissue factor mRNA.

Herpes simplex virus type I does not require productive infection to induce tissue factor in human umbilical vein endothelial cells.

Background: Herpes simplex virus (HSV)-infected endothelium is a model for vascular injury and possibly the development of atherosclerosis. In vitro infection of human umbilical vein endothelial cells (HUVEC) by HSV-1 results in a number of changes including the expression of a procoagulant activity (PCA) compatible with that due to tissue factor (TF) synthesis. In this study, we have further characterized this PCA using more stringent assays for TF, and examined whether virus rendered incapable of replication retains the ability to stimulate TF synthesis in HUVEC.

Functional neurokinin 1 receptors for substance P are expressed by human vascular endothelium.

Substance P (SP), a neurotachykinin, is important in a number of inflammatory processes in which the endothelial cell also plays a critical role. SP receptors have previously been identified only on arterial endothelium, and the scant in vitro evidence for direct effects of SP on human endothelium is based on studies using nonarterial cells. To better understand SP's role in inflammation, we sought to identify functional SP receptors on human endothelium in situ and in culture.

Herpes virus infection of endothelium: new insights into atherosclerosis.

Several pieces of evidence suggest that vascular endothelium may be a site of latent herpetic viral infection, and that activation of such infection might cause or aggravate atherosclerosis. The present studies which utilized HSV-1 infection of cultured endothelial monolayers, provide insights into two phenomena seemingly relevant in considerations of atherosclerosis. Thus, mechanisms are reported by which infected endothelium may be damaged by marginated inflammatory cells, and be transformed from an anticoagulant to a procoagulant tissue.

Hydrogen peroxide alters signal transduction in human endothelial cells.

Lytic H2O2-induced injury to human umbilical vein endothelial cells provides a model for endothelial cell damage in diverse states including acute respiratory distress and septic shock. Endothelial cell lysis is an extreme result of inflammatory cell activation. Functional alterations such as responsiveness to endothelial cell agonists and eicosanoid production might be impaired by exposure to inflammatory cell products including H2O2.

Infection of vascular endothelial cells with herpes simplex virus enhances tissue factor activity and reduces thrombomodulin expression.

Latent infection of vascular cells with herpes-viruses may play a pathogenic role in the development of human atherosclerosis. In a previous study, we found that cultured human umbilical vein endothelial cells (HUVECs) infected with herpes simplex virus 1 (HSV-1) became procoagulant, exemplified both by their enhanced assembly of the prothrombinase complex and by their inability to reduce adhesion of platelets. We now report two further procoagulant consequences of endothelial HSV infection: loss of surface thrombomodulin (TM) activity and induction of synthesis of tissue factor.

Endothelial cell platelet-activating factor primes neutrophil responses: amplification of endothelial activation by neutrophil products.

We have shown that platelet-activating factor (PAF) primes neutrophil (PMN) responses and enhances their ability to damage endothelial cells. Furthermore, thrombin-stimulated endothelial cells which produce PAF can augment and prime PMN superoxide production, elastase release and adhesion to endothelium. We wondered whether these marginated neutrophils (PMN) themselves, or their release products, might feedback and further amplify endothelial cell activation.

Measurement of intracellular calcium concentration in intact monolayers of human endothelial cells.

Intracellular calcium ([Ca++]i) plays an important role in signal transduction and cell activation. The measurement of [Ca++]i in intact monolayers of human umbilical vein endothelial cells with the fluorescent calcium-sensitive probe fura 2 has been evaluated. Monolayers provide a more physiologic cell preparation than suspensions and allow a greater variety of experimental manipulation.

Endothelial cell heterogeneity: antioxidant profiles determine vulnerability to oxidant injury.

Human umbilical vein endothelial cells were more sensitive to hydrogen peroxide lysis than cow pulmonary artery endothelial cells. Conversely, activated neutrophils which utilize hydrogen peroxide-mediated cell cytotoxicity cell mechanisms were more toxic to the cow pulmonary artery cells. This discordance was not related to neutrophil adhesion to either cell type or cell passage number.

Pulmonary hypertension and edema induced by platelet-activating factor in isolated, perfused rat lungs are blocked by BN52021.

The experimental intravenous administration of platelet activating factor (PAF) induces pulmonary hypertension and directly or indirectly increases capillary permeability. Selective PAF antagonists BN52021 and L652-731 have been shown to inhibit the action of PAF in vitro and in vivo. Using a unique isolated perfused rat lung model, we measured the effect of these PAF antagonists on PAF-induced pulmonary hypertension and edema.

Interleukin 1 or endotoxin increases the release of von Willebrand factor from human endothelial cells.

von Willebrand factor (vWF), a large adhesive glycoprotein, is synthesized by vascular endothelial cells (EC). Plasma levels of vWF manifest a broad normal range, and are elevated during sepsis and in inflammatory states. Since the inflammatory mediator, interleukin 1 (IL1) and bacterial endotoxin (LPS) both initiate procoagulant changes in vascular endothelium, we investigated the effect of these substances on endothelial cell release and residual endothelial cell content of vWF-antigen (vWFAg).

Interleukin 1 stimulates endothelial cell tissue factor production and expression by a prostaglandin-independent mechanism.

Activation of coagulation occurs at inflammatory sites following the ingress of mononuclear cells, and may result from alterations in the vessel wall. Since the monokine, interleukin 1, initiates diverse responses to inflammation, its ability to enhance vascular procoagulant activity was studied. Interleukin 1-treated cultured human endothelial cells acquired elevated levels of the procoagulant, tissue factor.

Magnesium sulfate: rationale for its use in preeclampsia.

Preeclampsia is a disorder of pregnancy characterized clinically by hypertension, proteinuria, and edema and characterized pathologically in its late stages by widespread microvascular thrombi. There is evidence from a number of studies that production of prostacyclin (prostaglandin I2, PGI2), a potent vasodilator and inhibitor of platelet aggregation, is deficient in preeclamptic compared to normal pregnancy. Traditional therapy utilizes infusions of large amounts of MgSO4, but the physiologic basis for this is not clear.

Structural features of endotoxin required for stimulation of endothelial cell tissue factor production; exposure of preformed tissue factor after oxidant-mediated endothelial cell injury.

Cultured human umbilical vein endothelial cells synthesize the procoagulant, tissue factor, after exposure to bacterial endotoxin. Wild-type lipopolysaccharide from Escherichia coli 0127:B8 stimulates a five- to 20-fold increase in cellular tissue factor. Similarly, rough or incomplete lipopolysaccharide subunits from mutant bacterial strains, or lipid A prepared by mild acid hydrolysis of whole endotoxin, are also stimulatory.

Protection against lethal hyperoxia by tracheal insufflation of erythrocytes: role of red cell glutathione.

Intact erythrocytes placed into the tracheobronchial tree of hyperoxic rats dramatically improved their chances for survival. Over 70 percent of the animals so treated survived more than 12 days during continuous exposure to 95 percent oxygen, whereas all of the control animals died within 96 hours. Lungs from erythrocyte-protected rats showed almost none of the morphologic damage suffered by untreated animals.

Insufflated red cells protect lungs from hyperoxic damage: role of red cell glutathione in scavenging toxic O2 radicals.

Hyperoxia-mediated pulmonary damage may involve formation of toxic oxygen species such as superoxide, hydrogen peroxide, and hydroxyl radical. Intact red cells evidently scavenge these when insufflated in small numbers into the tracheobronchial tree of rats. Such manipulation protects hyperoxic rats for prolonged periods of time and preserves normal pulmonary histology.

Bacterial adherence to fibronectin and endothelial cells: a possible mechanism for bacterial tissue tropism.

In the pathophysiology of endocarditis, bacteria must initially adhere to the endothelial surface components of the cardiac valve before invasion and colonization. The attachment of bacteria to endothelial cell surfaces is dependent on surface characteristics of both the bacteria and the endothelial cell. Fibronectin, a glycoprotein produced by endothelial cells, binds to some bacteria but not all.

Inflamed fibronectin: an altered fibronectin enhances neutrophil adhesion.

Recent investigations have emphasized the role of activated granulocytes in mediating vascular endothelial injury in the pathogenesis of shock lung. In vitro studies have indicated that tight adherence of the neutrophil to the endothelium is crucial for the development of cellular injury. Fibronectin is critical to cell-to-substratum and cell-to-cell interactions.