Endothelial Cell Cortactin Phosphorylation by Src Contributes to Polymorphonuclear Leukocyte Transmigration In Vitro

The underlying mechanisms that regulate leukocyte transendothelial migration through the vascular endothelium remain unclear. Cortactin is a substrate of Src tyrosine kinases and a regulator of cytoskeletal dynamics. Previous studies demonstrated a role for Src phosphorylation of cortactin in clustering of E-selectin and intercellular cell adhesion molecule-1 around adherent leukocytes. In the current study, we used an in vitro flow model to investigate the role of Src-induced cortactin phosphorylation in endothelium during polymorphonuclear leukocyte (PMN) transmigration through human umbilical vein endothelium (HUVEC) monolayers preactivated with tumor necrosis factor-{alpha}. Inhibition of Src in HUVEC using Src kinase inhibitors PP2 and SU6656 reduced PMN transmigration by 45±8% and 36±6%, respectively. Live cell imaging of green fluorescent protein–tagged cortactin in HUVEC revealed redistribution of cortactin in the region surrounding transmigrating PMN. Knockdown of cortactin in HUVEC by small interfering RNA also impaired transmigration to a similar degree, and this phenotype was rescued by reexpression of wild-type cortactin. Analysis of the location of initial arrest and locomotion of PMN adherent to HUVEC demonstrated that inhibition of Src tyrosine kinases or pretreatment with cortactin small interfering RNA reduced PMN transmigration at endothelial cell-to-cell junctions and not adhesion. Tyrosine phosphorylation of cortactin was important for transmigration, because expression of a mutant, in which the tyrosine phosphorylation sites were mutated to phenylalanine (cortactin3F), failed to rescue PMN transmigration. Moreover, expression of cortactin3F alone partially blocked PMN transmigration. These data suggest a model whereby tyrosine phosphorylation of cortactin by Src family kinases regulates PMN transmigratio

Inflammation Determines the Pro-Adhesive Properties of High Extracellular D-Glucose in Human Endothelial Cells In Vitro and Rat Microvessels In Vivo

Background: Hyperglycemia is acknowledged as an independent risk factor for developing diabetes-associated atherosclerosis. At present, most therapeutic approaches are targeted at a tight glycemic control in diabetic patients, although this fails to prevent macrovascular complications of the disease. Indeed, it remains highly controversial whether or not the mere elevation of extracellular D-glucose can directly promote vascular inflammation, which favors early proatherosclerotic events. Methods and Findings: In the present work, increasing extracellular D-glucose from 5.5 to 22 mmol/L was neither sufficient to induce intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression, analyzed by flow cytometry, nor to promote leukocyte adhesion to human umbilical vein endothelial cells (HUVEC) in vitro, measured by flow chamber assays. Interestingly, the elevation of D-glucose levels potentiated ICAM-1 and VCAM-1 expression and leukocyte adhesion induced by a pro-inflammatory stimulus, such as interleukin (IL)-1b (5 ng/mL). In HUVEC, high D-glucose augmented the activation of extracellular signal-regulated kinase 1/2 (ERK 1/2) and nuclear transcription factor-kB (NF-kB) elicited by IL-1b, measured by Western blot and electromobility shift assay (EMSA), respectively, but had no effect by itself. Both ERK 1/2 and NF-kB were necessary for VCAM-1 expression, but not for ICAM-1 expression. In vivo, leukocyte trafficking was evaluated in the rat mesenteric microcirculation by intravital microscopy. In accordance with the in vitro data, the acute intraperitoneal injection of D-glucose increased leukocyte rolling flux, adhesion and migration, but only when IL-1b was coadministered. Conclusions: These results indicate that the elevation of extracellular D-glucose levels is not sufficient to promote vascular inflammation, and they highlight the pivotal role of a pro-inflammatory environment in diabetes, as a critical factor conditioning the early pro-atherosclerotic actions of hyperglycemia

KLF2 Is a Novel Transcriptional Regulator of Endothelial Proinflammatory Activation

The vascular endothelium is a critical regulator of vascular function. Diverse stimuli such as proinflammatory cytokines and hemodynamic forces modulate endothelial phenotype and thereby impact on the development of vascular disease states. Therefore, identification of the regulatory factors that mediate the effects of these stimuli on endothelial function is of considerable interest. Transcriptional profiling studies identified the Kruppel-like factor (KLF)2 as being inhibited by the inflammatory cytokine interleukin-1β and induced by laminar shear stress in cultured human umbilical vein endothelial cells. Overexpression of KLF2 in umbilical vein endothelial cells robustly induced endothelial nitric oxide synthase expression and total enzymatic activity. In addition, KLF2 overexpression potently inhibited the induction of vascular cell adhesion molecule-1 and endothelial adhesion molecule E-selectin in response to various proinflammatory cytokines. Consistent with these observations, in vitro flow assays demonstrate that T cell attachment and rolling are markedly attenuated in endothelial monolayers transduced with KLF2. Finally, our studies implicate recruitment by KLF2 of the transcriptional coactivator cyclic AMP response element–binding protein (CBP/p300) as a unifying mechanism for these various effects. These data implicate KLF2 as a novel regulator of endothelial activation in response to proinflammatory stimuli

Dynamic purine signaling and metabolism during neutrophil–endothelial interactions

During episodes of hypoxia and inflammation, polymorphonuclear leukocytes (PMN) move into underlying tissues by initially passing between endothelial cells that line the inner surface of blood vessels (transendothelial migration, TEM). TEM creates the potential for disturbances in vascular barrier and concomitant loss of extravascular fluid and resultant edema. Recent studies have demonstrated a crucial role for nucleotide metabolism and nucleoside signaling during inflammation. These studies have implicated multiple adenine nucleotides as endogenous tissue protective mechanisms invivo. Here, we review the functional components of vascular barrier, identify strategies for increasing nucleotide generation and nucleoside signaling, and discuss potential therapeutic targets to regulate the vascular barrier during inflammation

Regulation of adenine nucleotide concentration at endothelium-fluid interface by viscous shear flow.

The action of adenine nucleotides on vascular endothelial cells is apparently mediated by the local flow conditions. Because nucleotides are sequentially degraded from ATP-->ADP-->AMP-->adenosine by ecto-enzymes at the endothelial surface, it has been hypothesized that the observed flow effect is caused by the flow-dependent change of nucleotide concentration at the cell surface. In this study, we have calculated the concentration profiles of adenine nucleotides at the cell surface under flow conditions encountered in an in vitro parallel-plate flow system, as has been used in several related experimental studies. When medium containing uniformly distributed ATP is perfused over endothelial monolayers, our results show that ATP concentration in the cell vicinity gradually decreases in the streamwise direction as a result of enzymatic degradation. This hydrolysis of ATP results in the generation of ADP, and ADP concentration in turn gradually increases at the cell surface. The concentration profiles of nucleotides are dependent on the levels of applied wall shear rate. As the corresponding shear stress increases from 0.1 to 30 dynes/cm2, ATP concentration at the cell surface at the center of coverslip increases from 0.66 to 0.93. Under no-flow conditions, our model predicts a steady decline of ATP concentration and a transient increase of ATP-derived ADP, comparable to the published results of previous experiments. These numerical results, combined with our recent experimental data, provide insights into the cellular mechanisms by which hemodynamic flow modulates the effects of vasoactive agents on endothelium

Monocyte attachment to activated human vascular endothelium in vitro is mediated by leukocyte adhesion molecule-1 (L-selectin) under nonstatic conditions

The receptors that mediate monocyte adhesion to cytokine-stimulated endothelial monolayers were assessed using a nonstatic (rotating) cell-attachment assay. In this system, leukocyte adhesion molecule-1 (LAM-1) (L-selectin) mediated a major portion (87 +/- 15% at 37 degrees C) of monocyte attachment to activated endothelium. mAb blocking of endothelial leukocyte adhesion molecule-1 (41% inhibition), CD18 (36%), and vascular cell adhesion molecule-1 (25%) function had lesser effects on attachment. These results suggest that LAM-1 may serve an important role in monocyte attachment to endothelium at sites of inflammation