Biomedical subjects

Dennis F Kucik

Publications and source records attributed to Dennis F Kucik.

10 recordsLinked to original sources

PubMed2006

Intracellular heterogeneity in adhesiveness of endothelium affects early steps in leukocyte adhesion.

Endothelial cell junctions are thought to be preferential sites for transmigration. However, the factors that determine the site of transmigration are not well defined. Our data show that the preferential role of endothelial cell junctions is not limited to transmigration but extends to earlier steps of leukocyte recruitment, such as rolling and arrest. We used primary mouse neutrophils and mouse aortic endothelium in a flow chamber system to compare adhesive interactions near endothelial cell junctions to interactions over endothelial cell centers. We found differences in both rolling velocity and arrest frequency for neutrophils at endothelial cell junctions vs. more central areas of endothelial cells. Differences were governed by adhesion molecule interactions, not local topography. Interestingly, the role of particular adhesion molecules depended on their location on the endothelial cell surface. Although ICAM-1 stabilized and slowed rolling over central areas of the cell, it did not influence rolling velocity over endothelial cell junctions. P-selectin and VCAM-1 were more important for rolling near endothelial cell junctions than E-selectin. This demonstrates that adhesive properties of endothelial cell junctions influence early events in the adhesion cascade, which may help explain how leukocytes are localized to sites of eventual transmigration.

Animals↗

PubMed2005

Revealing anti-inflammatory mechanisms of soy isoflavones by flow: modulation of leukocyte-endothelial cell interactions.

The antiatherogenic effects of soy isoflavone consumption have been demonstrated in a variety of studies. However, the mechanisms involved remain poorly defined. Adhesion of monocytes to vascular endothelial cells is a key step within the inflammatory cascade that leads to atherogenesis. Many factors, including the physical forces associated with blood flow, regulate this process. Using an in vitro flow assay, we report that genistein, a principal component of most isoflavone preparations, inhibits monocyte adhesion to cytokine (TNF-alpha)-stimulated human vascular endothelial cells at physiologically relevant concentrations (0-1 microM). This effect is absolutely dependent on flow and is not observed under static conditions. Furthermore, this inhibition was dependent on activation of endothelial peroxisomal proliferator-activated receptor-gamma. No significant role for other reported properties of genistein, including antioxidant effects, inhibition of tyrosine kinases, or activation of estrogen receptors, was observed. Furthermore, the antiadhesive effects of genistein did not occur via modulation of the adhesion molecules E-selectin, ICAM-1, VCAM-1, or platelet-endothelial cell adhesion molecule-1. These data reveal a novel anti-inflammatory mechanism for isoflavones and identify the physical forces associated with blood flow and a critical mediator of this function.

Anti-Inflammatory Agents↗

PubMed2005

Both Th1 and Th2 cells require P-selectin glycoprotein ligand-1 for optimal rolling on inflamed endothelium.

The acquisition of homing receptors that redirect lymphocyte trafficking to nonlymphoid tissues after antigen encounter is a fundamental aspect of effector T-cell development. Although a role for selectins and their ligands has been well characterized for trafficking of Th1 cells to nonlymphoid sites, mechanisms responsible for Th2 trafficking are not well understood. Using a flow chamber system in which the endothelial interactions of two distinct T-cell populations could be examined simultaneously, we directly compared the requirements for Th1 and Th2 cell tethering and rolling. We found that although Th2 cells expressed significantly lower levels of selectin ligands than Th1 cells, activation of the endothelium by Th2-derived factors induced rolling interactions that were comparable for both Th1 and Th2 populations. Further, in the absence of PSGL-1, no other adhesion molecule could effectively compensate for lack of PSGL-1 to mediate rolling of either Th1 or Th2 cells. Thus, both Th1 and Th2 populations express functional PSGL-1-based selectin ligands for tethering and rolling on activated endothelium, and both effector populations can use PSGL-1 as the dominant scaffold for functional selectin ligand expression.

Animals↗

PubMed2005

Cell-adhesion assays.

One of the most important properties of cells that are derived from multicellular organisms is their ability to adhere to extracellular matrix proteins or other cells. Analysis of cell-extracellular matrix and/or cell-cell adhesion, therefore, is of important value to experimental biologists as well as clinical investigators. Over the past several decades, many different cell-adhesion assays have been developed. Based on the experimental conditions, most of the cell-adhesion assays fall into two categories, namely static adhesion assays and flow adhesion assays. Static assays are widely used to assess the adhesion of many types of cells (e.g., epithelial cells and fibroblasts) to the extracellular matrix. The flow adhesion assays are more appropriate for analysis of blood cell (e.g., leukocyte) adhesion to endothelial cells, to each other, or extracellular matrix proteins. This chapter describes two basic protocols, one for analysis of cell adhesion under static conditions and the other for measurement of cell adhesion under shear stress. In addition, variations to the basic protocols and areas where special attention is required for successful application of these methods are discussed.

Animals↗

PubMed2004

Intercellular adhesion molecule-1 (ICAM-1) regulates endothelial cell motility through a nitric oxide-dependent pathway.

Coordinated regulation of endothelial cell migration is an integral process during angiogenesis. However, molecular mechanisms regulating endothelial cell migration remain largely unknown. Increased expression of cell adhesion molecules has been implicated during angiogenesis, yet the precise role of these molecules is unclear. Here, we examined the hypothesis that intercellular adhesion molecule-1 (ICAM-1) is important for endothelial cell migration. Total cell displacement and directional migration were significantly attenuated in ICAM-1-deficient endothelium. Closer examination of ICAM-1-deficient cells revealed decreased Akt Thr(308) and endothelial nitric-oxide synthase Ser(1177) phosphorylation and NO bioavailability, increased actin stress fiber formation, and a lack of distinct cell polarity compared with wild-type endothelium. Supplementation of ICAM-1 mutant cells with the NO donor DETA NONOate (0.1 microM) corrected the migration defect, diminished stress fiber formation, and enhanced pseudopod and uropod formation. These data demonstrate that ICAM-1 facilitates the development of cell polarity and modulates endothelial cell migration through a pathway regulating endothelial nitric-oxide synthase activation and organization of the actin cytoskeleton.

Animals↗

PubMed2003

Thrombospondin signaling through the calreticulin/LDL receptor-related protein co-complex stimulates random and directed cell migration.

The matricellular extracellular matrix protein thrombospondin-1 (TSP1) stimulates focal adhesion disassembly through a sequence (known as the hep I peptide) in its heparin-binding domain. This mediates signaling through a receptor co-complex involving calreticulin and low-density lipoprotein (LDL) receptor-related protein (LRP). We postulate that this transition to an intermediate adhesive state enhances cellular responses to dynamic environmental conditions. Since cell adhesion dynamics affect cell motility, we asked whether TSP1/hep I-induced intermediate adhesion alters cell migration. Using both transwell and Dunn chamber assays, we demonstrate that TSP1 and hep I gradients stimulate endothelial cell chemotaxis. Treatment with focal adhesion-labilizing concentrations of TSP1/hep I in the absence of a gradient enhances endothelial cell random migration, or chemokinesis, associated with an increase in cells migrating, migration speed, and total cellular displacement. Calreticulin-null and LRP-null fibroblasts do not migrate in response to TSP1/hep I, nor do endothelial cells treated with the LRP inhibitor receptor-associated protein (RAP). Furthermore, TSP1/hep I-induced focal adhesion disassembly is associated with reduced chemotaxis to basic fibroblast growth factor (bFGF) but enhanced chemotaxis to acidic (a)FGF, suggesting differential modulation of growth factor-induced migration. Thus, TSP1/hep I stimulation of intermediate adhesion regulates the migratory phenotype of endothelial cells and fibroblasts, suggesting a role for TSP1 in remodeling responses.

Animals↗

PubMed2003

High-temporal-resolution analysis demonstrates that ICAM-1 stabilizes WEHI 274.1 monocytic cell rolling on endothelium.

Leukocyte rolling, adhesion, and migration on vascular endothelium involve several sets of adhesion molecules that interact simultaneously. Each of these receptor-ligand pairs may play multiple roles. We examined the role of ICAM-1 in adhesive interactions with mouse aortic endothelial cells (MAECs) in an in vitro flow system. Average rolling velocity of the monocytic cell line WEHI 274.1 was increased on ICAM-1-deficient MAECs compared with wild-type MAECs, both with and without TNF-alpha stimulation. High-temporal-resolution analysis provided insights into the underlying basis for these differences. Without TNF-alpha stimulation, average rolling velocity was slower on wild-type than on ICAM-1-deficient endothelium because of brief (<1 s) pauses. On TNF-alpha-stimulated ICAM-1-deficient endothelium, cells rolled faster because of transient accelerations, producing "jerky" rolling. Firm adhesion to ICAM-1-deficient MAECs was significantly reduced compared with wild-type MAECs, although the number of rolling cells was similar. These results demonstrate directly that ICAM-1 affects rolling velocity by stabilizing leukocyte rolling.

Animals↗

PubMed2003

Avidity modulation activates adhesion under flow and requires cooperativity among adhesion receptors.

An early step in activation of leukocyte adhesion is a release of integrins from cytoskeletal constraints on their diffusion, leading to rearrangement and, consequently, increased avidity. Static adhesion assays using purified ligand as a substrate have demonstrated that very low doses of cytochalasin D disconnect beta2-integrins from their cytoskeletal links, allowing rearrangement and activating adhesion. The adhesion process in blood vessels is poorly simulated by these assays, however, for two reasons: leukocyte adhesion to endothelium 1), occurs in the presence of blood flow and 2), involves the simultaneous interactions of multiple sets of adhesion molecules. We investigated the effect of cytochalasin D, at concentrations that increase integrin diffusion but do not alter leukocyte shape and surface features, on adhesion of leukocytes to endothelial cells under flow. Cytochalasin D increased the number of rolling cells, the number of firmly adherent cells, and the duration of both rolling and firm adhesion. These effects required endothelial cell expression of ICAM-1, the ligand for leukocyte beta2-integrins. The beta2-integrin-ICAM-1 interaction alone was not sufficient, however. Experiments using purified substrates demonstrated that avidity effects on activation of adhesion under flow require functional cooperativity between integrins and other adhesion receptors.

Animals↗

PubMed2002

Rearrangement of integrins in avidity regulation by leukocytes.

Leukocyte adhesion must be tightly controlled in order for leukocytes to patrol the body as nonadherent cells, yet stop and emigrate from the blood into tissues at sites of infection or inflammation. A key element in this process is activation of beta2 integrins. While beta2 integrin activation involves conformational changes that increase affinity for ligand, evidence is accumulating that rearrangement of integrins, resulting in increases in avidity, is at least as important in regulating binding capacity. Recent work has established the importance of diffusion and rearrangement of integrins to activation of leukocyte adhesion, and has begun to unravel the molecular basis of its regulation.

Animals↗

PubMed2001

Inhibition of phosphatidylinositol 3-kinase enhances mitogenic actions of insulin in endothelial cells.

The concept of "selective insulin resistance" has emerged as a unifying hypothesis in attempts to reconcile the influence of insulin resistance with that of hyperinsulinemia in the pathogenesis of macrovascular complications of diabetes. To explore this hypothesis in endothelial cells, we designed a set of experiments to mimic the "typical metabolic insulin resistance" by blocking the phosphatidylinositol 3-kinase pathway and exposing the cells to increasing concentrations of insulin ("compensatory hyperinsulinemia"). Inhibition of phosphatidylinositol 3-kinase with wortmannin blocked the ability of insulin to stimulate increased expression of endothelial nitric-oxide synthase, did not affect insulin-induced activation of MAP kinase, and increased the effects of insulin on prenylation of Ras and Rho proteins. At the same time, this experimental paradigm resulted in increased expression of vascular cellular adhesion molecules-1 and E-selectin, as well as increased rolling interactions of monocytes with endothelial cells. We conclude that inhibition of the metabolic branch of insulin signaling leads to an enhanced mitogenic action of insulin in endothelial cells.

Androstadienes↗