Biomedical subjects

Michael A Gimbrone

Publications and source records attributed to Michael A Gimbrone.

12 recordsLinked to original sources

PubMed2004

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. Two prototypic arterial waveforms, "athero-prone" and "athero-protective," were defined as representative of the wall shear stresses in two distinct regions of the carotid artery (carotid sinus and distal internal carotid artery) that are typically "susceptible" or "resistant," respectively, to atherosclerotic lesion development. These two waveforms were applied to cultured EC, and cDNA microarrays were used to analyze the differential patterns of EC gene expression. In addition, the differential effects of athero-prone vs. athero-protective waveforms were further characterized on several parameters of EC structure and function, including actin cytoskeletal organization, expression and localization of junctional proteins, activation of the NF-kappaB transcriptional pathway, and expression of proinflammatory cytokines and adhesion molecules. These global gene expression patterns and functional data reveal a distinct phenotypic modulation in response to the wall shear stresses present in atherosclerosis-susceptible vs. atherosclerosis-resistant human arterial geometries.

Arteriosclerosis↗

PubMed2004

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-1beta 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.

Base Sequence↗

PubMed2004

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., N = 3), standard statistical methods such as the Student's t-test fail to produce reliable results. Two other statistical approaches commonly used to improve significance estimates are a penalized t-test and a Z-test using intensity-dependent variance estimates. RESULTS: The performance of these approaches is compared using a dataset of 23 replicates, and a new implementation of the Z-test is introduced that pools together variance estimates of genes with similar minimum intensity. Significance estimates based on 3 replicate arrays are calculated using each statistical technique, and their accuracy is evaluated by comparing them to a reliable estimate based on the remaining 20 replicates. The reproducibility of each test statistic is evaluated by applying it to multiple, independent sets of 3 replicate arrays. Two implementations of a Z-test using intensity-dependent variance produce more reproducible results than two implementations of a penalized t-test. Furthermore, the minimum intensity-based Z-statistic demonstrates higher accuracy and higher or equal precision than all other statistical techniques tested. CONCLUSION: An intensity-based variance estimation technique provides one simple, effective approach that can improve p-value estimates for differentially regulated genes derived from replicated microarray datasets. Implementations of the Z-test algorithms are available at http://vessels.bwh.harvard.edu/software/papers/bmcg2004.

DNA, Complementary↗

PubMed2004

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. Expression of neuronal NOS and inducible NOS was not detectable in cells of either genotype. Upon stimulation, confluent wild-type MLECs produced significant amounts of NO compared with N(omega)-monomethyl-l-arginine-treated wild-type cells. eNOS knockout and wild-type cells showed no difference in the expression of E-selectin, P-selectin, intracellular adhesion molecule-1, and vascular cell adhesion molecule-1 as measured by flow cytometry on the surface of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31)-positive cells. Both eNOS knockout and wild-type cells displayed the characteristics of resting endothelium. Adhesion studies in a parallel plate laminar flow chamber showed no difference in leukocyte-endothelial cell interactions between the two genotypes. Cytokine treatment induced endothelial cell adhesion molecule expression and increased leukocyte-endothelial cell interactions in both genotypes. We conclude that in resting murine endothelial cells, absence of endothelial production of NO by itself does not initiate endothelial cell activation or promote leukocyte-endothelial cell interactions. We propose that eNOS derived NO does not chronically suppress endothelial cell activation in an autocrine fashion but serves to counterbalance signals that mediate activation.

Animals↗

PubMed2004

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. The hemodynamic characteristics of the arterialized vein, as assessed by ultrasound and magnetic resonance imaging, resemble features of the arterial circulation. Temporal analyses of the morphological changes in the venous segment at 1, 3, and 7 days after surgery demonstrate preservation of the endothelium at all time points and formation of multiple smooth muscle layers by day 7. Expression of endothelial E-selectin and VCAM-1 was documented at early time points, concomitant with the presence of neutrophils and monocytes/macrophages in the vascular wall. In addition, endothelium-dependent permeability was decreased in the arterialized vein when compared to the contralateral control vein. Thus, this novel mouse model of venous arterialization displays anatomical and cellular features present in other species, and should help to characterize the molecular mechanisms of this adaptive response of the vascular wall to changes in its biomechanical environment.

Animals↗

PubMed2003

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. In this study, we show that E-selectin is localized in cholesterol-rich lipid rafts at the cell surface, and that upon ligation E-selectin clusters and redistributes in the plasma membrane colocalizing with a fraction of caveolin-1-containing rafts. In addition, we demonstrate that leukocyte adhesion via E-selectin results in association with and activation of phospholipase Cgamma (PLCgamma). Moreover, we show that disruption of lipid rafts with the cholesterol-depleting drug methyl-beta-cyclodextrin disrupts the raft localization of E-selectin as well as the ligation-induced association of E-selectin with PLCgamma, and subsequent tyrosine phosphorylation of PLCgamma. In contrast, cholesterol depletion has no effect on E-selectin-dependent mitogen-activated protein kinase activation. Thus, these findings demonstrate that the presence of E-selectin in lipid rafts is necessary for its association with, and activation of, PLCgamma, and suggest that this subcellular localization of E-selectin is related to its signaling function(s) during leukocyte-endothelial interactions.

Antibodies, Monoclonal↗

PubMed2003

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. Despite two major advances in treatment, low VT ventilation for ALI/ARDS and activated protein C for severe sepsis (the leading cause of ALI/ARDS), additional research is needed to develop specific treatments and improve understanding of the pathogenesis of these syndromes. The NHLBI convened a working group to develop specific recommendations for future ALI/ARDS research. Improved understanding of disease heterogeneity through use of evolving biologic, genomic, and genetic approaches should provide major new insights into pathogenesis of ALI. Cellular and molecular methods combined with animal and clinical studies should lead to further progress in the detection and treatment of this complex disease.

Academies and Institutes↗

PubMed2003

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). Our study revealed that only MHECs exhibited high constitutive VCAM-1 expression. Exposure to TNF-alpha up-regulated adhesion molecule expression and chemokine production in both MLECs and MHECs. However, high levels of Regulated on Activation Normal T cell Expressed And Secreted (RANTES) expression were detected only in TNF-alpha-activated MHECs. TNF-alpha-stimulated MLECs and MHECs both supported T-helper cell interactions under defined flow conditions. Most T cells instantaneously arrested on MHECs but exhibited a rolling phenotype on MLECs. Blocking studies revealed that T-cell arrest on MHECs was mediated by constitutive VCAM-1 and TNF-alpha-induced RANTES. These findings are consistent with the hypothesis that functional heterogeneity of endothelial cells from different sites exists and some of it is retained in vitro. Furthermore, these results provide an insight into the molecular mechanisms that may mediate T-helper cell recruitment to these organs.

Animals↗

PubMed2002

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). PGE(2) (50 nm) attenuated LPS-induced mRNA and protein expression of chemokines including monocyte chemoattractant protein-1, interleukin-8, macrophage inflammatory protein-1alpha and -1beta, and interferon-inducible protein-10. PGE(2) also inhibited the tumor necrosis factor-alpha-, interferon-gamma-, and interleukin-1beta-mediated expression of these chemokines. In contrast to the case of MPhi, PGE(2) did not suppress chemokine expression in human endothelial and smooth muscle cells (SMC) treated with LPS and pro-inflammatory cytokines. To assess the potential paracrine effect of endogenous PGE(2) on macrophage-derived chemokine production, we co-cultured MPhi with SMC in the presence of LPS. In these co-cultures, cyclooxygenase-2-dependent PGE(2) production exceeded that in the mono-cultures, and MIP-1beta declined significantly compared with MPhi cultured without SMC. We further documented prominent expression of the PGE(2) receptor EP4 in MPhi in both culture and human atheroma. Moreover, a selective EP4 antagonist completely reversed PGE(2)-mediated suppression of chemokine production. Thus, endogenous PGE(2) may modulate inflammation during atherogenesis and other inflammatory diseases by suppressing macrophage-derived chemokine production via the EP4 receptor.

Anti-Inflammatory Agents↗

PubMed2002

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. Because induction of both VCAM-1 and E-selectin by these cytokines is NF-kappaB dependent, we investigated whether the inhibitory effect of hyperosmotic medium might involve IRF-1. Electrophoretic mobility shift assays of the VCAM-1 promoter demonstrated that hyperosmotic medium suppressed IL-1beta- or TNF-alpha-activated binding activities of IRF-1, but not NF-kappaB, to their respective sites. Hyperosmotic medium also inhibited the expression of IRF-1 induced by TNF-alpha or IFN-gamma. Furthermore, hyperosmotic medium inhibited TNF-alpha or IFN-gamma induction of guanylate binding protein-1, another IRF-1-dependent gene. Taken together, hyperosmolarity selectively inhibits cytokine-induced VCAM-1 in endothelial cells, via an IRF-1-dependent mechanism. Thus, pathophysiological fluctuations in plasma osmolarity may influence certain endothelial-dependent components of the inflammatory response and host defense mechanisms.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed2002

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. To test whether IKK beta is necessary for cytokine activation of human vascular endothelium and endothelial recruitment of human monocytes under laminar flow, we constructed a recombinant adenoviral vector carrying a dominant negative mutant of IKK beta (Ad.dnIKK beta) to transduce human umbilical vein endothelial cells (HUVEC) in vitro. We found that dnIKK beta expression effectively blocked NF-kappa B activation as assessed by nuclear translocation of NF-kappa B, I kappa B degradation, and NF-kappa B dependent reporter expression, without affecting activation of the other relevant signaling pathways, SAPK/JNK and p38. Furthermore, overexpression of dnIKK beta in TNF-alpha-stimulated HUVEC blocked induction of the surface adhesion molecules E-selectin, ICAM-1, and VCAM-1. Under simulated physiologic flow conditions, both firm adhesion and rolling of human peripheral monocytes on dnIKK beta-transduced endothelial monolayers were markedly inhibited. We conclude that IKK beta is necessary for the cytokine-induced inflammatory phenotype of human endothelium and endothelial recruitment of human monocytes under flow.

Adenoviridae↗

PubMed2002

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. Arterial pulsatile flow for 24 hours was compared to an equivalent time-average steady laminar shear stress, using no flow (static) culture conditions as a baseline. While both flow stimuli induced comparable changes in cell shape and alignment, distinct patterns of responses were observed in the distribution of actin stress fibers and vinculin-associated adhesion complexes, intrinsic migratory characteristics, and the expression of eNOS mRNA and protein. These results thus reveal a unique responsiveness of ECs to an arterial waveform and begin to elucidate the complex sensing capabilities of the endothelium to the dynamic characteristics of flows throughout the human vascular tree.

Adaptation, Physiological↗