Keith R. Anderson

Functional profiling of the Saccharomyces cerevisiae genome

Determining the effect of gene deletion is a fundamental approach to understanding gene function. Conventional genetic screens exhibit biases, and genes contributing to a phenotype are often missed. We systematically constructed a nearly complete collection of gene-deletion mutants (96% of annotated open reading frames, or ORFs) of the yeast Saccharomyces cerevisiae. DNA sequences dubbed ‘molecular bar codes’ uniquely identify each strain, enabling their growth to be analysed in parallel and the fitness contribution of each gene to be quantitatively assessed by hybridization to high-density oligonucleotide arrays. We show that previously known and new genes are necessary for optimal growth under six well-studied conditions: high salt, sorbitol, galactose, pH 8, minimal medium and nystatin treatment. Less than 7% of genes that exhibit a significant increase in messenger RNA expression are also required for optimal growth in four of the tested conditions. Our results validate the yeast gene-deletion collection as a valuable resource for functional genomics.

Endothelial Dysfunction, Hemodynamic Forces, and Atherogenesisa

Abstract: 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 NFκB 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‐1β). 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.

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.

Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling.

Intracellular lipopolysaccharide from Gram-negative bacteria including Escherichia coli, Salmonella typhimurium, Shigella flexneri, and Burkholderia thailandensis activates mouse caspase-11, causing pyroptotic cell death, interleukin-1β processing, and lethal septic shock. How caspase-11 executes these downstream signalling events is largely unknown. Here we show that gasdermin D is essential for caspase-11-dependent pyroptosis and interleukin-1β maturation. A forward genetic screen with ethyl-N-nitrosourea-mutagenized mice links Gsdmd to the intracellular lipopolysaccharide response. Macrophages from Gsdmd−/− mice generated by gene targeting also exhibit defective pyroptosis and interleukin-1β secretion induced by cytoplasmic lipopolysaccharide or Gram-negative bacteria. In addition, Gsdmd−/− mice are protected from a lethal dose of lipopolysaccharide. Mechanistically, caspase-11 cleaves gasdermin D, and the resulting amino-terminal fragment promotes both pyroptosis and NLRP3-dependent activation of caspase-1 in a cell-intrinsic manner. Our data identify gasdermin D as a critical target of caspase-11 and a key mediator of the host response against Gram-negative bacteria.

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-β (TGF-β) 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-β 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-β-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-β 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.

Endothelial Gene Regulation by Laminar Shear Stress

Endothelial cells, because of their unique localization, are constantly exposed to fluid mechanical forces derived by the flowing blood. These forces, and more specifically shear stresses; affect endothelial structure and function, both in vivo and in vitro, and are implicated as contributing factors in the development of cardiovascular diseases. We have demonstrated earlier that the shear stress selectively induces the transcription of several endothelial genes, and have defined a shear stress response element (SSRE) in the promoter of platelet-derived-growth-factor B (PDGF-B), that is shared by additional endothelial shear stress responsive genes. Here we further characterize this SSRE and the nuclear factors that bind to it, and imply the possible role of the endothelium cytoskeleton in transducing shear stress, leading to the expression of PDGF-B/SSRE constructs in transfected endothelial cells exposed to shear stress. We also present, yet a new shear stress response element in the Platelet Derived Growth Factor A promoter, that contains a binding site to the transcription factors egr1/sp1. These results further demonstrate the complexity of gene regulation by hemodynamic forces, and support the important part that these forces have in the physiology and pathophysiology of the vessel wall.

Mechanosensitive Endothelial Gene Expression Profiles

Abstract: The possibility that hemodynamic forces can act as a “local risk factor” for endothelial dysfunction provides a conceptual framework for the long‐standing 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.

Expression of the bumetanide-sensitive Na-K-Cl cotransporter BSC2 is differentially regulated by fluid mechanical and inflammatory cytokine stimuli in vascular endothelium.

In vascular endothelium, the electroneutral Na-K-Cl cotransport system is thought to function in the maintenance of a selective permeability barrier in certain vascular beds (e.g., brain), as well as in the preservation of endothelial homeostasis in the face of fluctuating osmotic conditions that may accompany certain pathophysiological conditions (e.g., diabetes mellitus). Here we demonstrate that the gene encoding the bumetanide-sensitive cotransporter BSC2, one of the two major isoforms of Na-K-Cl cotransporters present in mammalian cells, can be differentially regulated by inflammatory cytokines and fluid mechanical forces in cultured endothelium. Interleukin-1beta and tumor necrosis factor-alpha significantly upregulate expression of BSC2 mRNA and protein in human umbilical vein endothelial cells, a response that is inhibited by pretreatment with interferon-gamma. Steady laminar fluid shear stress, at a physiologic magnitude (10 dyn/cm2), is also able to induce and maintain elevated expression of BSC2 in cultured human umbilical vein endothelial cells, while a comparable time-averaged magnitude of turbulent fluid shear stress is not. In vivo, BSC2 mRNA is upregulated after intraperitoneal administration of bacterial endotoxin (LPS) in murine lung and kidney, but not in cardiac tissue. These results provide the first experimental evidence that the BSC2 gene can be selectively regulated by different inflammatory cytokine and fluid mechanical stimuli in endothelium, and support a role for BSC2 in vascular homeostasis and inflammation.

The Critical Role of Mechanical Forces in Blood Vessel Development, Physiology and Pathology

The following extended abstracts were presented at the Research Initiatives in Vascular Disease Conference, Movers and Shakers in the Vascular Tree-Hemodynamic and Biomechanical Factors in Blood Vessel Pathology, sponsored by The Lifeline Foundation and the Cardiovascular & Interventional Radiology Research and Educational Foundation; jointly sponsored by the International Society for Cardiovascular Surgery, North American Chapter, The Society for Vascular Surgery, and The Society of Cardiovascular and Interventional Radiology; in cooperation with the National Institutes of Health-National Heart, Lung &Blood Institute on Mar 11-12, 1999, in Bethesda, Md.

Human Prostaglandin Transporter Gene (hPGT) is Regulated by Fluid Mechanical Stimuli in Cultured Endothelial Cells and Expressed in Vascular Endothelium in Vivo

BACKGROUND biomechanical forces generated by blood flow within the cardiovascular system have been proposed as important modulators of regional endothelial phenotype and function. This process is thought to involve the regulation of vascular gene expression by physiological fluid mechanical stimuli such as fluid shear stresses. METHODS AND RESULTS We demonstrate sustained upregulation of a recently identified gene encoding a human prostaglandin transporter (hPGT) in cultured human vascular endothelium exposed to a physiological fluid mechanical stimulus in vitro. This biomechanical induction is selective in that steady laminar shear stress is sufficient to upregulate the hPGT gene at the level of transcriptional activation, whereas a comparable level of turbulent shear stress (a nonphysiological stimulus) is not. Various biochemical stimuli, such as bacterial endotoxin and the inflammatory cytokines recombinant human interleukin 1beta cytokines (rhIL-1beta) and tumor necrosis factor-alpha (TNF-alpha), did not significantly induce hPGT. Using a specific antiserum to hPGT, we demonstrate endothelial expression within the arterial vasculature and the microcirculation of highly vascularized tissues such as the heart. CONCLUSIONS Our results identify hPGT as an inducible gene in vascular endothelium and suggest that biomechanical stimuli generated by blood flow in vivo may be important determinants of hPGT expression. Furthermore, this demonstration of regulated endothelial expression of hPGT implicates this molecule in the regional metabolism of prostanoids within the cardiovascular system.