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.

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. All of these downstream signaling events appear to require an intact cytoplasmic domain of E-selectin. Here we demonstrate that tyrosine 603 in the cytoplasmic domain of E-selectin is required for the E-selectin-dependent ERK1/2 activation. Tyrosine 603 plays an important role in mediating the association of E-selectin with SHP2, and the catalytic domain of SHP2 is, in turn, critical for E-selectin-dependent ERK1/2 activation. An adapter protein complex consisting of Shc.Grb2.Sos bridges between SHP2 and the Ras.Raf.phospho-MEK macrocomplex. These molecular events thus outline a mechanism by which cross-linking of E-selectin by engagement of ligands on adherent leukocytes can initiate a multifunctional signaling pathway in the activated endothelial cell at sites of inflammation.

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. Argus imports the intensities and images of externally quantified microarray spots, performs normalization, and calculates ratios of gene expression between conditions. The database can be queried locally or over the Web, providing a convenient format for Web-publishing entire microarray data sets. Searches for regulated genes can be conducted across multiple experiments, and the integrated results incorporate images of the actual hybridization spots for artifact screening. Query results are presented in a clone- or gene-oriented fashion to rapidly identify highly regulated genes, and scatterplots of expression ratios allow an individual ratio to be interpreted in the context of all data points in the experiment. Algorithms were developed to optimize response times for queries of regulated genes. Supporting databases are updated easily to maintain current gene identity information, and hyperlinks to the Web provide access to descriptions of gene function. Query results also can be exported for higher-order analyses of expression patterns. This combination of features currently is not available in similar software. Argus is available at http://vessels.bwh.harvard.edu/software/Argus.

Hmg-CoA reductase inhibitor modulates monocyte-endothelial cell interaction under physiological flow conditions in vitro: involvement of Rho GTPase-dependent mechanism.

3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, or statins, have been reported to exert actions independent of their lipid-lowering effects. To critically assess the effects of statins on monocyte-endothelial cell interactions, we used an in vitro model that mimicked physiological flow conditions. Monocytic U937 cells were incubated in the presence of cerivastatin for 48 hours. Adhesive interactions of statin-treated U937 cells were then analyzed by use of activated (interleukin-1beta 10 U/mL, 4 hours) human umbilical vein endothelial cells in an in vitro flow apparatus. Flow cytometric analysis of adhesion molecules and measurement of F-actin content in U937 cells were performed before and after statin treatment. Preincubation with cerivastatin significantly decreased U937 firm adhesion to activated human umbilical vein endothelial cells, whereas U937 rolling was not decreased. Fluorescence-activated cell sorter analysis revealed downregulation of U937 surface expression of CD11a, CD18, and VLA4 after statin treatment. Cerivastatin significantly reduced F-actin content in U937 cells and inhibited RhoA translocation, whereas preincubation with C3 exoenzyme reduced U937 adhesion under flow. Cerivastatin reduces monocyte adhesion to vascular endothelium under physiological flow conditions via downregulation of integrin adhesion molecules and inhibition of actin polymerization via RhoA inactivation. Our findings have important implications for the lipid-independent effects of statins.

Biomechanical activation of vascular endothelium as a determinant of its functional phenotype.

One of the striking features of vascular endothelium, the single-cell-thick lining of the cardiovascular system, is its phenotypic plasticity. Various pathophysiologic factors, such as cytokines, growth factors, hormones, and metabolic products, can modulate its functional phenotype in health and disease. In addition to these humoral stimuli, endothelial cells respond to their biomechanical environment, although the functional implications of this biomechanical paradigm of activation have not been fully explored. Here we describe a high-throughput genomic analysis of modulation of gene expression observed in cultured human endothelial cells exposed to two well defined biomechanical stimuli-a steady laminar shear stress and a turbulent shear stress of equivalent spatial and temporal average intensity. Comparison of the transcriptional activity of 11,397 unique genes revealed distinctive patterns of up- and down-regulation associated with each type of stimulus. Cluster analyses of transcriptional profiling data were coupled with other molecular and cell biological techniques to examine whether these global patterns of biomechanical activation are translated into distinct functional phenotypes. Confocal immunofluorescence microscopy of structural and contractile proteins revealed the formation of a complex apical cytoskeleton in response to laminar shear stress. Cell cycle analysis documented different effects of laminar and turbulent shear stresses on cell proliferation. Thus, endothelial cells have the capacity to discriminate among specific biomechanical forces and to translate these input stimuli into distinctive phenotypes. The demonstration that hemodynamically derived stimuli can be strong modulators of endothelial gene expression has important implications for our understanding of the mechanisms of vascular homeostasis and atherogenesis.

Expression of ephrinB2 identifies a stable genetic difference between arterial and venous vascular smooth muscle as well as endothelial cells, and marks subsets of microvessels at sites of adult neovascularization.

The transmembrane ligand ephrinB2 and its receptor tyrosine kinase EphB4 are molecular markers of embryonic arterial and venous endothelial cells, respectively, and are essential for angiogenesis. Here we show that expression of ephrinB2 persists in adult arteries where it extends into some of the smallest diameter microvessels, challenging the classical view that capillaries have neither arterial nor venous identity. EphrinB2 also identifies arterial microvessels in several settings of adult neovascularization, including tumor angiogenesis, contravening the dogma that tumor vessels arise exclusively from postcapillary venules. Unexpectedly, expression of ephrinB2 also defines a stable genetic difference between arterial and venous vascular smooth muscle cells. These observations argue for revisions of classical concepts of capillary identity and the topography of neovascularization. They also imply that ephrinB2 may be functionally important in neovascularization and in arterial smooth muscle, as well as in embryonic angiogenesis.

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. To address this fundamental question, we undertook high-throughput transcriptional profiling to assess the global patterns of gene expression in cultured endothelial cells exposed to two defined biomechanical stimuli. Analyses of the transcriptional activity of thousands of genes have revealed unique patterns of gene expression associated with certain types of stimuli. These unique gene expression programs and their associated functional phenotypes constitute the strongest evidence to date that vascular endothelial cells can discriminate among different types of biomechanical stimuli. The results of these studies and the working hypotheses inspired by detailed molecular analyses of biomechanically activated vascular endothelium promise to provide new insights into the role of hemodynamics in the pathogenesis of atherosclerosis.

Adenoviral transduction of human E-selectin into isolated, perfused, rat aortic segments: an ex vivo model for studying leukocyte-endothelial interactions.

E-selectin, a member of the selectin family of adhesion molecules, is thought to play an important role in leukocyte-endothelial (EC) interactions during inflammation and atherosclerosis. To critically examine the role of E-selectin in leukocyte-EC interactions in the vascular system, we created a recombinant adenoviral vector containing a human E-selectin cDNA (AdRSVE-sel) and examined the effect of AdRSVE-sel in an ex vivo vascular model of a rat aortic segment. A segment of abdominal aorta was isolated from a male Sprague-Dawley rat transduced with AdRSVE-sel ex vivo. After 72 h, surface expression of transduced E-selectin in the segment was confirmed by Western blotting and immunohistochemistry using anti-E-selectin mAb. Aortic segments were connected to a perfusion system and the adhesion of human polymorphonuclear neutrophils (PMN), and a human monocytic cell line (THP-1) to the EC surface was studied in the presence of a physiological level of flow (0.85 ml/min, approximate luminal surface shear stress=1.76 dyn/cm2). Adhesion of PMN was assessed by scanning electron microscopy and quantified using fluorescently labeled PMN. AdRSVE-sel transduced aortic segments mediated significantly more PMN and THP-1 adhesion than control segments transduced with AdRSVLacZ. Pretreatment of AdRSVE-sel transduced aortic segments with anti-E-selectin mAb inhibited PMN adhesion significantly, as well as THP-1. These data indicate that human E-selectin expressed in rat aortic segments can support the adhesion of human PMN as well as THP-1 under physiological flow conditions. This genetically modified, excised, vascular-segment model provides a useful tool for the study of leukocyte recruitment in the vascular system.

Inhibition of E-selectin gene expression by transforming growth factor beta in endothelial cells involves coactivator integration of Smad and nuclear factor kappaB-mediated signals.

Transforming growth factor (TGF)-beta(1) is a pleiotropic cytokine/growth factor that is thought to play a critical role in the modulation of inflammatory events. We demonstrate that exogenous TGF-beta(1) can inhibit the expression of the proinflammatory adhesion molecule, E-selectin, in vascular endothelium exposed to inflammatory stimuli both in vitro and in vivo. This inhibitory effect occurs at the level of transcription of the E-selectin gene and is dependent on the action of Smad proteins, a class of intracellular signaling proteins involved in mediating the cellular effects of TGF-beta(1). Furthermore, we demonstrate that these Smad-mediated effects in endothelial cells result from a novel competitive interaction between Smad proteins activated by TGF-beta(1) and nuclear factor kappaB (NFkappaB) proteins activated by inflammatory stimuli (such as cytokines or bacterial lipopolysaccharide) that is mediated by the transcriptional coactivator cyclic AMP response element-binding protein (CREB)-binding protein (CBP). Augmentation of the limited amount of CBP present in endothelial cells (via overexpression) or selective disruption of Smad-CBP interactions (via a dominant negative strategy) effectively antagonizes the ability of TGF-beta(1) to block proinflammatory E-selectin expression. These data thus demonstrate a novel mechanism of interaction between TGF-beta(1)-regulated Smad proteins and NFkappaB proteins regulated by inflammatory stimuli in vascular endothelial cells. This type of signaling mechanism may play an important role in the immunomodulatory actions of this cytokine/growth factor in the cardiovascular system.

E-selectin-dependent signaling via the mitogen-activated protein kinase pathway in vascular endothelial cells.

E-selectin, a cytokine-inducible adhesion molecule, supports rolling and stable arrest of leukocytes on activated vascular endothelium. Previous studies have suggested that this transmembrane protein can also transduce signals into the endothelial cell. We now demonstrate activation of the mitogen-activated protein kinase (MAPK) signaling cascade in cultured HUVEC in response to E-selectin-dependent leukocyte adhesion and Ab-mediated cross-linking of cell surface E-selectin. Adhesion of increasing numbers of HL60 cells to IL-1beta-activated HUVEC stimulated robust increases in MAPK activity that were abrogated by an E-selectin blocking Ab. Cross-linking of cell surface E-selectin with Abs, as a mimic of multivalent ligand engagement, strongly stimulated MAPK/extracellular signal-related kinase (ERK) kinase (MEK)-dependent MAPK activation and concomitant up-regulation of mRNA for c-fos, an immediate early response gene, whereas Ab cross-linking of HLA class I molecules (present at comparable density) failed to do so. Coimmunoprecipitation documented Ras, Raf-1 and, phospho-MEK complex formation. Unactivated HUVEC transduced with a full-length adenoviral E-selectin construct also exhibited cross-link-induced MAPK activation, macromolecular complex formation, and c-fos up-regulation, whereas HUVEC transduced with a cytoplasmic domain deletion mutant failed to respond. These observations indicate that E-selectin can transduce an activating stimulus via the MAPK cascade into the endothelial cell during leukocyte adhesion.

Endothelial dysfunction, hemodynamic forces, and atherogenesis.

Phenotypic modulation of endothelium to a dysfunctional state contributes to the pathogenesis of cardiovascular diseases such as atherosclerosis. The localization of atherosclerotic lesions to arterial geometries associated with disturbed flow patterns suggests an important role for local hemodynamic forces in atherogenesis. 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 stimuli and transduce them into genetic regulatory events. At the level of individual genes, this regulation is accomplished via the binding of certain transcription factors, such as NF kappa B and Egr-1, to shear-stress response elements (SSREs) that are present in the promoters of biomechanically inducible genes. At the level of multiple genes, distinct patterns of up- and downregulation appear to be elicited by exposure to steady laminar shear stresses versus comparable levels of non-laminar (e.g., turbulent) shear stresses or cytokine stimulation (e.g., IL-1 beta). Certain genes upregulated by steady laminar shear stress stimulation (such as eNOS, COX-2, and Mn-SOD) support vasoprotective (i.e., anti-inflammatory, anti-thrombotic, anti-oxidant) functions in the endothelium. We hypothesize that the selective and sustained expression of these and related "atheroprotective genes" in the endothelial lining of lesion-protected areas represents a mechanism whereby hemodynamic forces can influence lesion formation and progression.

C-C and C-X-C chemokines trigger firm adhesion of monocytes to vascular endothelium under flow conditions.

In summary, our findings indicate that specific chemokines that are elaborated by endothelial cells after cytokine or endotoxin activation can play an essential role in monocyte recruitment beyond their chemoattractant activities. We show that this action is to translate initial monocyte tethering into firm adhesion via rapid leukocyte integrin activation. The in vitro model presented here provides a sensitive system for investigating the modulating ability of chemokines and reveals an important biological effect that is not predicted by results in simpler in vitro assays, such as measurement of calcium transients or chemotaxis. The surprising finding that the C-X-C chemokine IL-8 can trigger monocyte firm adhesion to vascular endothelium suggests a potential role for this chemokine in monocyte recruitment and underscores the biological complexity of the chemokine family.

A role for smad6 in development and homeostasis of the cardiovascular system.

Smad proteins are intracellular mediators of signalling initiated by Tgf-betasuperfamily ligands (Tgf-betas, activins and bone morphogenetic proteins (Bmps)). Smads 1, 2, 3, 5 and 8 are activated upon phosphorylation by specific type I receptors, and associate with the common partner Smad4 to trigger transcriptional responses. The inhibitory Smads (6 and 7) are transcriptionally induced in cultured cells treated with Tgf-beta superfamily ligands, and downregulate signalling in in vitro assays. Gene disruption in mice has begun to reveal specific developmental and physiological functions of the signal-transducing Smads. Here we explore the role of an inhibitory Smad in vivo by targeted mutation of Madh6 (which encodes the Smad6 protein). Targeted insertion of a LacZ reporter demonstrated that Smad6 expression is largely restricted to the heart and blood vessels, and that Madh6 mutants have multiple cardiovascular abnormalities. Hyperplasia of the cardiac valves and outflow tract septation defects indicate a function for Smad6 in the regulation of endocardial cushion transformation. The role of Smad6 in the homeostasis of the adult cardiovascular system is indicated by the development of aortic ossification and elevated blood pressure in viable mutants. These defects highlight the importance of Smad6 in the tissue-specific modulation of Tgf-beta superfamily signalling pathways in vivo.

Vascular endothelial cells respond to spatial gradients in fluid shear stress by enhanced activation of transcription factors.

The vascular endothelium is exposed to a spectrum of fluid mechanical forces generated by blood flow; some of these, such as fluid shear stress, can directly modulate endothelial gene expression. Previous work by others and in our laboratory, using an in vitro uniform laminar shear stress model, has identified various shear stress response elements (SSREs) within the promoters of certain endothelial genes that regulate their expression by interacting with various transcription factors, including nuclear factor-kappaB (NF-kappaB), early growth response-1 (Egr-1), and activator protein-1 (AP-1, composed of c-Jun/c-Jun and c-Jun/c-Fos protein dimers). In the current study, we have examined the topographical patterns of NF-kappaB, Egr-1, c-Jun, and c-Fos activation in a specially designed in vitro disturbed laminar shear stress model, which incorporates regions of significant spatial shear stress gradients similar to those found in atherosclerosis-prone arterial geometries in vivo (eg, arterial bifurcations, curvatures, ostial openings). Using newly developed quantitative image analysis techniques, we demonstrate that endothelial cells subjected to disturbed laminar shear stress exhibit increased levels of nuclear localized NF-kappaB, Egr-1, c-Jun, and c-Fos, compared with cells exposed to uniform laminar shear stress or maintained under static conditions. In addition, individual cells display a heterogeneity in responsiveness to disturbed flow, as measured by the amount of NF-kappaB, Egr-1, c-Jun, and c-Fos in their nuclei. This differential regulation of transcription factor expression by disturbed versus uniform laminar shear stress indicates that regional differences in blood flow patterns in vivo-in particular, the occurrence of spatial shear stress gradients-may represent important local modulators of endothelial gene expression at anatomic sites predisposed for atherosclerotic development.

MCP-1 and IL-8 trigger firm adhesion of monocytes to vascular endothelium under flow conditions.

Monocytes contribute to the development of atherosclerotic lesions in mouse models. The chemoattractant proteins (chemokines), monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8), are found in human atheroma, and mice lacking receptors for these chemokines are less susceptible to atherosclerosis and have fewer monocytes in vascular lesions. Although MCP-1 has a powerful effect on monocytes, IL-8 is thought to act predominantly on neutrophils and it is unclear how it could recruit monocytes. Here we investigate the ability of chemokines to control the interaction of monocytes under flow conditions with vascular endothelium that has been transduced to express specific leukocyte-adherence receptors. We find that MCP-1 and IL-8 can each rapidly cause rolling monocytes to adhere firmly onto monolayers expressing E-selectin, whereas related chemokines do not. These effects do not correlate with either the induction of a calcium transient or chemotaxis. We conclude that chemokines are important modulators of monocyte-endothelial interactions under flow conditions. Moreover, our finding that IL-8 is a powerful trigger for firm adhesion of monocytes to vascular endothelium reveals an unexpected role for this chemokine in monocyte recruitment.

MEKK-1, a component of the stress (stress-activated protein kinase/c-Jun N-terminal kinase) pathway, can selectively activate Smad2-mediated transcriptional activation in endothelial cells.

Smad proteins are essential components of the intracellular signaling pathways utilized by members of the transforming growth factor-beta (TGF-beta) superfamily of growth factors. Certain Smad proteins (e.g. Smad1, -2, and -3) can act as regulated transcriptional activators, a process that involves phosphorylation of these proteins by activated TGF-beta superfamily receptors. We demonstrate that the intracellular kinase mitogen-activated protein kinase kinase kinase-1 (MEKK-1), an upstream activator of the stress-activated protein kinase/c-Jun N-terminal kinase pathway, can participate in Smad2-dependent transcriptional events in cultured endothelial cells. A constitutively active form of MEKK-1 but not mitogen-activated protein kinase kinase-1 (MEK-1) or TGF-beta-activated kinase-1, two distinct intracellular kinases, can specifically activate a Gal4-Smad2 fusion protein, and this effect correlates with an increase in the phosphorylation state of the Smad2 protein. These effects do not require the presence of the C-terminal SSXS motif of Smad2 that is the site of TGF-beta type 1 receptor-mediated phosphorylation. Activation of Smad2 by active MEKK-1 results in enhanced Smad2-Smad4 interactions, nuclear localization of Smad2 and Smad4, and the stimulation of Smad protein-transcriptional coactivator interactions in endothelial cells. Overexpression of Smad7 can inhibit the MEKK-1-mediated stimulation of Smad2 transcriptional activity. A physiological level of fluid shear stress, a known activator of endogenous MEKK-1 activity in endothelial cells, can stimulate Smad2-mediated transcriptional activity. These data demonstrate a novel mechanism for activation of Smad protein-mediated signaling in endothelial cells and suggest that Smad2 may act as an integrator of diverse stimuli in these cells.