Endothelial Cell Dysfunction and the Pathobiology of Atherosclerosis.

Dysfunction of the endothelial lining of lesion-prone areas of the arterial vasculature is an important contributor to the pathobiology of atherosclerotic cardiovascular disease. Endothelial cell dysfunction, in its broadest sense, encompasses a constellation of various nonadaptive alterations in functional phenotype, which have important implications for the regulation of hemostasis and thrombosis, local vascular tone and redox balance, and the orchestration of acute and chronic inflammatory reactions within the arterial wall. In this review, we trace the evolution of the concept of endothelial cell dysfunction, focusing on recent insights into the cellular and molecular mechanisms that underlie its pivotal roles in atherosclerotic lesion initiation and progression; explore its relationship to classic, as well as more recently defined, clinical risk factors for atherosclerotic cardiovascular disease; consider current approaches to the clinical assessment of endothelial cell dysfunction; and outline some promising new directions for its early detection and treatment.

Vascular endothelium, hemodynamics, and the pathobiology of atherosclerosis.

The localization of atherosclerotic lesion formation to regions of disturbed blood flow associated with certain arterial geometries, in humans and experimental animals, suggests an important role for hemodynamic forces in the pathobiology of atherosclerosis. There is increasing evidence that the vascular endothelium, which is directly exposed to various fluid mechanical forces generated by pulsatile blood flow, can discriminate among these different biomechanical stimuli and transduce them into genetic regulatory programs that modulate endothelial function. In this brief review, we discuss how biomechanical stimuli generated by blood flow can influence endothelial functional phenotypes, and explore the working hypothesis of "atheroprone" hemodynamic environments as "local risk factors" in atherogenesis.

The Gordon Wilson lecture. Understanding vascular endothelium: a pilgrim's progress. Endothelial dysfunction, biomechanical forces and the pathobiology of atherosclerosis.

These are exciting times for the biomedical sciences, in general, and, in particular, for those who strive to understand the origins of complex human diseases, as we begin to focus with increasing precision on disease mechanisms at the cellular and molecular levels. Armed with the high-through-put technologies of the Post-Genomic Era, we now face the challenge of understanding biological systems at the level of their complex integration, and this will truly bring meaning to the concept of Systems Biology.

Biomechanical forces in atherosclerosis-resistant vascular regions regulate endothelial redox balance via phosphoinositol 3-kinase/Akt-dependent activation of Nrf2.

Local patterns of biomechanical forces experienced by endothelial cells (ECs) in different vascular geometries appear to play an essential role in regulating EC function and determining the regional susceptibility to atherosclerosis, even in the face of systemic risk factors. To study how biomechanical forces regulate EC redox homeostasis, an important pathogenic factor in atherogenesis, we have cultured human ECs under 2 prototypic arterial shear stress waveforms, "atheroprone" and "atheroprotective," which were derived from 2 distinct vascular regions in vivo that are typically "susceptible" or "resistant" to atherosclerosis. We demonstrate that atheroprotective flow decreases EC intracellular redox level and protects ECs against oxidative stress-induced injury.

Biomechanical modulation of endothelial phenotype: implications for health and disease.

The functional phenotypic plasticity of the vascular endothelium relies on the ability of individual endothelial cells to integrate and transduce both humoral and biomechanical stimuli from their surrounding environments. Increasing evidence strongly suggests that biomechanical stimulation is a critical determinant of endothelial gene expression and the functional phenotypes displayed by these cells in several pathophysiological conditions. Herein we discuss the types of biomechanical forces that endothelial cells are constantly exposed to within the vasculature, explain how these biomechanical stimuli serve as regulators of endothelial function and discuss the increasing evidence that "atherosclerosis-protective" or "atherosclerosis-prone" haemodynamic environments can be important causative factors for atherogenesis via the differential regulation of endothelial transcriptional programmes.

Integration of flow-dependent endothelial phenotypes by Kruppel-like factor 2.

In the face of systemic risk factors, certain regions of the arterial vasculature remain relatively resistant to the development of atherosclerotic lesions. The biomechanically distinct environments in these arterial geometries exert a protective influence via certain key functions of the endothelial lining; however, the mechanisms underlying the coordinated regulation of specific mechano-activated transcriptional programs leading to distinct endothelial functional phenotypes have remained elusive. Here, we show that the transcription factor Kruppel-like factor 2 (KLF2) is selectively induced in endothelial cells exposed to a biomechanical stimulus characteristic of atheroprotected regions of the human carotid and that this flow-mediated increase in expression occurs via a MEK5/ERK5/MEF2 signaling pathway.

Statins exert endothelial atheroprotective effects via the KLF2 transcription factor.

3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, statins, have been shown to positively impact vascular function independent of their plasma lipid-lowering action. Several of these beneficial effects involve modulation of gene expression. Here we explored whether the transcription factor Kruppel-like factor 2 (KLF2), a biomechanically activated gene we recently identified as part of the endothelial "atheroprotective phenotype," is regulated by statins and whether this mechanism is important for the non-lipid lowering beneficial effects mediated by these drugs in endothelium.

Kruppel-like factor 2 (KLF2) regulates endothelial thrombotic function.

The vascular endothelium maintains blood fluidity by inhibiting blood coagulation, inhibiting platelet aggregation, and promoting fibrinolysis. Endothelial cells lose these nonthrombogenic properties on exposure to proinflammatory stimuli. We recently identified the Kruppel-like factor KLF2 as a novel regulator of endothelial proinflammatory activation.

Distinct endothelial phenotypes evoked by arterial waveforms derived from atherosclerosis-susceptible and -resistant regions of human vasculature.

Atherosclerotic lesion localization to regions of disturbed flow within certain arterial geometries, in humans and experimental animals, suggests an important role for local hemodynamic forces in atherogenesis. To explore how endothelial cells (EC) acquire functional/dysfunctional phenotypes in response to vascular region-specific flow patterns, we have used an in vitro dynamic flow system to accurately reproduce arterial shear stress waveforms on cultured human EC and have examined the effects on EC gene expression by using a high-throughput transcriptional profiling approach. The flow patterns in the carotid artery bifurcations of several normal human subjects were characterized by using 3D flow analysis based on actual vascular geometries and blood flow profiles.

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.

Improving the statistical detection of regulated genes from microarray data using intensity-based variance estimation.

Background: Gene microarray technology provides the ability to study the regulation of thousands of genes simultaneously, but its potential is limited without an estimate of the statistical significance of the observed changes in gene expression. Due to the large number of genes being tested and the comparatively small number of array replicates (e.g.

Role of endothelial nitric oxide synthase in endothelial activation: insights from eNOS knockout endothelial cells.

The objective of this study was to determine whether absence of endothelial nitric oxide synthase (eNOS) affects the expression of cell surface adhesion molecules in endothelial cells. Murine lung endothelial cells (MLECs) were prepared by immunomagnetic bead selection from wild-type and eNOS knockout mice. Wild-type cells expressed eNOS, but eNOS knockout cells did not.

Early adaptive responses of the vascular wall during venous arterialization in mice.

Venous arterialization occurs when a vein segment is transposed as a bypass graft into the arterial circulation, resulting in a structural and functional reorganization of the vascular wall in response to the new local biomechanical environment. Although the anatomical changes of venous arterialization have been well characterized, the molecular mechanisms of vascular remodeling remain incompletely understood. Here, we present a novel model of venous arterialization in mice wherein the external jugular vein is connected to the common carotid artery.

Lipid raft localization of cell surface E-selectin is required for ligation-induced activation of phospholipase C gamma.

E-selectin, an endothelial cell surface adhesion receptor for leukocytes, also acts as a signaling receptor. Upon multivalent ligation, E-selectin transduces outside-in signals into the endothelium leading to changes in intracellular Ca(2+) concentration and activation of the mitogen-activated protein kinase signaling pathway. In addition, following leukocyte engagement, E-selectin associates via its cytoplasmic domain with components of the actin cytoskeleton and undergoes alterations in phosphorylation state that result in changes in gene expression.

Heterogeneity of endothelial cells from different organ sites in T-cell subset recruitment.

Chemokines and adhesion molecules play a critical role in the recruitment of leukocytes into specific organ sites. Little is known, however, regarding the repertoire of chemokines and adhesion molecules expressed within different vascular beds. In this study, we compare adhesion molecule expression, chemokine induction, and T-cell subset-endothelial interactions under defined flow conditions on resting and tumor necrosis factor (TNF)-alpha-activated murine lung endothelial cells (MLECs) and heart endothelial cells (MHECs).

Future research directions in acute lung injury: summary of a National Heart, Lung, and Blood Institute working group.

Acute lung injury (ALI) and its more severe form, the acute respiratory distress syndrome (ARDS), are syndromes of acute respiratory failure that result from acute pulmonary edema and inflammation. The development of ALI/ARDS is associated with several clinical disorders including direct pulmonary injury from pneumonia and aspiration as well as indirect pulmonary injury from trauma, sepsis, and other disorders such as acute pancreatitis and drug overdose. Although mortality from ALI/ARDS has decreased in the last decade, it remains high.

Prostaglandin E2 suppresses chemokine production in human macrophages through the EP4 receptor.

Pro-inflammatory pathways participate in the pathogenesis of atherosclerosis. However, the role of endogenous anti-inflammatory pathways in atheroma has received much less attention. Therefore, using cDNA microarrays, we screened for genes regulated by prostaglandin E(2) (PGE(2)), a potential endogenous anti-inflammatory mediator, in lipopolysaccharide (LPS)-treated human macrophages (MPhi).

A new in vitro model to evaluate differential responses of endothelial cells to simulated arterial shear stress waveforms.

In the circulation, flow-responsive endothelial cells (ECs) lining the lumen of blood vessels are continuously exposed to complex hemodynamic forces. To increase our understanding of EC response to these dynamic shearing forces, a novel in vitro flow model was developed to simulate pulsatile shear stress waveforms encountered by the endothelium in the arterial circulation. A modified waveform modeled after flow patterns in the human abdominal aorta was used to evaluate the biological responsiveness of human umbilical vein ECs to this new type of stimulus.

Hyperosmotic stimuli inhibit VCAM-1 expression in cultured endothelial cells via effects on interferon regulatory factor-1 expression and activity.

Transcriptional up-regulation of the VCAM-1 gene, induced by proinflammatory cytokines such as IL-1beta and TNF-alpha, requires activation of not only NF-kappaB, but also involves interferon regulatory factor (IRF)-1. During a study of gene induction by mechanical stimuli in cultured human endothelial cells, we noted that medium hyperosmolarity appeared to influence cytokine-induced expression of VCAM-1. Indeed, addition of hyperosmotic, pathophysiologically relevant concentrations of NaCl effectively inhibited IL-1beta or TNF-alpha induction of VCAM-1, but not E-selectin, at the level of mRNA and cell surface protein.

Endothelial IKK beta signaling is required for monocyte adhesion under laminar flow conditions.

Endothelial activation induces expression of pro-inflammatory molecules that are thought to play an important role in atherogenesis through enhanced vascular monocyte recruitment. Many pro-inflammatory endothelial signals are transcriptionally regulated by members of the NF- kappa B family. The serine-threonine kinase, IKK beta, can mediate NF- kappa B activation although several alternative pathways exist.

Mechanosensitive endothelial gene expression profiles: scripts for the role of hemodynamics in atherogenesis?

The possibility that hemodynamic forces can act as a "local risk factor" for endothelial dysfunction provides a conceptual framework for the longstanding observation that the earliest lesions of atherosclerosis develop in a nonrandom pattern, the geometries of which correlate with branch points and other regions of altered blood flow. This has led us to hypothesize that hemodynamic forces, in particular wall shear stresses generated by complex patterns of blood flow, can function as both positive and negative stimuli in atherogenesis via effects on endothelial cell gene expression. To understand how endothelial cells in different regions of the arterial tree acquire both functional and dysfunctional phenotypes due to regional hemodynamics, it was important to begin to delineate, in a comprehensive fashion, the mechanoresponsiveness of endothelial cells.

Molecular events in transmembrane signaling via E-selectin. SHP2 association, adaptor protein complex formation and ERK1/2 activation.

E-selectin is a cytokine-inducible adhesion molecule that is expressed by activated endothelial cells at sites of inflammation. In addition to supporting rolling and stable arrest of leukocytes, there is increasing evidence that E-selectin functions in transmembrane signaling into endothelial cells during these adhesive interactions. We have previously shown that adhesion of HL-60 cells (which express ligands for E-selectin), or antibody-mediated cross-linking of E-selectin, results in formation of a Ras/Raf-1/phospho-MEK macrocomplex, extracellular signal-regulated protein kinase (ERK1/2) activation, and c-fos up-regulation.

Argus--a new database system for Web-based analysis of multiple microarray data sets.

The ongoing revolution in microarray technology allows biologists studying gene expression to routinely collect >10(5) data points in a given experiment. Widely accessible and versatile database software is required to process this large amount of raw data into a format that facilitates the development of new biological insights. Here, we present a novel microarray database software system, named Argus, designed to process, analyze, manage, and publish microarray data.