Bench-to-bedside review: Future novel diagnostics for sepsis - a systems biology approach

The early, accurate diagnosis and risk stratification of sepsis remains an important challenge in the critically ill. Since traditional biomarker strategies have not yielded a gold standard marker for sepsis, focus is shifting towards novel strategies that improve assessment capabilities. The combination of technological advancements and information generated through the human genome project positions systems biology at the forefront of biomarker discovery. While previously available, developments in the technologies focusing on DNA, gene expression, gene regulatory mechanisms, protein and metabolite discovery have made these tools more feasible to implement and less costly, and they have taken on an enhanced capacity such that they are ripe for utilization as tools to advance our knowledge and clinical research. Medicine is in a genome-level era that can leverage the assessment of thousands of molecular signals beyond simply measuring selected circulating proteins. Genomics is the study of the entire complement of genetic material of an individual. Epigenetics is the regulation of gene activity by reversible modifications of the DNA. Transcriptomics is the quantification of the relative levels of messenger RNA for a large number of genes in specific cells or tissues to measure differences in the expression levels of different genes, and the utilization of patterns of differential gene expression to characterize different biological states of a tissue. Proteomics is the large-scale study of proteins. Metabolomics is the study of the small molecule profiles that are the terminal downstream products of the genome and consists of the total complement of all low-molecular-weight molecules that cellular processes leave behind. Taken together, these individual fields of study may be linked during a systems biology approach. There remains a valuable opportunity to deploy these technologies further in human research. The techniques described in this paper not only have the potential to increase the spectrum of diagnostic and prognostic biomarkers in sepsis, but they may also enable the discovery of new disease pathways. This may in turn lead us to improved therapeutic targets. The objective of this paper is to provide an overview and basic framework for clinicians and clinical researchers to better understand the 'omics technologies' to enhance further use of these valuable tools

Functional Vascular Endothelium Derived from Human Induced Pluripotent Stem Cells

Summary Vascular endothelium is a dynamic cellular interface that displays a unique phenotypic plasticity. This plasticity is critical for vascular function and when dysregulated is pathogenic in several diseases. Human genotype-phenotype studies of endothelium are limited by the unavailability of patient-specific endothelial cells. To establish a cellular platform for studying endothelial biology, we have generated vascular endothelium from human induced pluripotent stem cells (iPSCs) exhibiting the rich functional phenotypic plasticity of mature primary vascular endothelium. These endothelial cells respond to diverse proinflammatory stimuli, adopting an activated phenotype including leukocyte adhesion molecule expression, cytokine production, and support for leukocyte transmigration. They maintain dynamic barrier properties responsive to multiple vascular permeability factors. Importantly, biomechanical or pharmacological stimuli can induce pathophysiologically relevant atheroprotective or atheroprone phenotypes. Our results demonstrate that iPSC-derived endothelium possesses a repertoire of functional phenotypic plasticity and is amenable to cell-based assays probing endothelial contributions to inflammatory and cardiovascular diseases

Elevated levels of placental growth factor represent an adaptive host response in sepsis

Recently, we demonstrated that circulating levels of vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) are increased in sepsis (Yano, K., P.C. Liaw, J.M. Mullington, S.C. Shih, H. Okada, N. Bodyak, P.M. Kang, L. Toltl, B. Belikoff, J. Buras, et al. 2006. J. Exp. Med. 203:1447–1458). Moreover, enhanced VEGF/Flk-1 signaling was shown to contribute to sepsis morbidity and mortality. We tested the hypothesis that PlGF also contributes to sepsis outcome. In mouse models of endotoxemia and cecal ligation puncture, the genetic absence of PlGF or the systemic administration of neutralizing anti-PlGF antibodies resulted in higher mortality compared with wild-type or immunoglobulin G–injected controls, respectively. The increased mortality associated with genetic deficiency of PlGF was reversed by adenovirus (Ad)-mediated overexpression of PlGF. In the endotoxemia model, PlGF deficiency was associated with elevated circulating levels of VEGF, induction of VEGF expression in the liver, impaired cardiac function, and organ-specific accentuation of barrier dysfunction and inflammation. Mortality of endotoxemic PlGF-deficient mice was increased by Ad-mediated overexpression of VEGF and was blocked by expression of soluble Flt-1. Collectively, these data suggest that up-regulation of PlGF in sepsis is an adaptive host response that exerts its benefit, at least in part, by attenuating VEGF signaling

Endothelial cells present antigens in vivo

BACKGROUND: Immune recognition of vascular endothelial cells (EC) has been implicated in allograft rejection, protection against pathogens, and lymphocyte recruitment. However, EC pervade nearly all tissues and predominate in none, complicating any direct test of immune recognition. Here, we examined antigen presentation by EC in vivo by testing immune responses against E. coli β-galactosidase (β-gal) in two lines of transgenic mice that express β-gal exclusively in their EC. TIE2-lacZ mice express β-gal in all EC and VWF-lacZ mice express β-gal in heart and brain microvascular EC. RESULTS: Transgenic and congenic wild type FVB mice immunized with β-gal expression vector DNA or β-gal protein generated high titer, high affinity antisera containing comparable levels of antigen-specific IgG1 and IgG2a isotypes, suggesting equivalent activation of T helper cell subsets. The immunized transgenic mice remained healthy, their EC continued to express β-gal, and their blood vessels showed no histological abnormalities. In response to β-gal in vitro, CD4(+ )and CD8(+ )T cells from immunized transgenic and FVB mice proliferated, expressed CD25, and secreted IFN-γ. Infection with recombinant vaccinia virus encoding β-gal raised equivalent responses in transgenic and FVB mice. Hearts transplanted from transgenic mice into FVB mice continued to beat and the graft EC continued to express β-gal. These results suggested immunological ignorance of the transgene encoded EC protein. However, skin transplanted from TIE2-lacZ onto FVB mice lost β-gal(+ )EC and the hosts developed β-gal-specific antisera, demonstrating activation of host immune effector mechanisms. In contrast, skin grafted from TIE2-lacZ onto VWF-lacZ mice retained β-gal(+ )EC and no antisera developed, suggesting a tolerant host immune system. CONCLUSION: Resting, β-gal(+ )EC in transgenic mice tolerize specific lymphocytes that would otherwise respond against β-gal expressed by EC within transplanted skin. We conclude that EC effectively present intracellular "self" proteins to the immune system. However, antigen presentation by EC does not delete or anergize a large population of specific lymphocytes that respond to the same protein following conventional immunization with protein or expression vector DNA. These results clearly demonstrate striking context sensitivity in the immune recognition of EC, a subtlety that must be better understood in order to treat immune diseases and complications involving the vasculature

Preconditioning of primary human endothelial cells with inflammatory mediators alters the “set point” of the cell

Endothelial cells are highly sensitive to changes in the extracellular milieu. Sepsis results in activation of inflammatory and coagulation pathways. We hypothesized that sepsis-associated mediators may alter the response capacity (so-called “set point”) of endothelial cells. Human umbilical vein endothelial cells (HUVEC) were preincubated in the presence or absence of tumor necrosis factor (TNF)-α, lipopolysaccharide (LPS), hypoxia, hyperthermia, and/or high glucose; treated with or without thrombin for 4 h; and then processed for RNase protection assays of selected activation markers. Priming with TNF-α and LPS significantly inhibited thrombin-mediated induction of vascular cell adhesion molecule-1, intercellular adhesion molecule-1, tissue factor, and E-selectin, but not platelet-derived growth factor-A or CD44. In electrophoretic mobility shift assays, thrombin-treated HUVEC demonstrated inducible binding of p65 NF-κB, an effect that was significantly blunted by pretreatment of cells with TNF-α and LPS. Consistent with these results, TNF-α and LPS attenuated the effect of thrombin on IκB phosphorylation, total cytoplasmic IκB, and nuclear translocation of p65 NF-κB. The inhibitory effect of TNF-α on thrombin signaling persisted for up to 24 h following removal of the cytokine. Taken together, these data suggest that inflammatory mediators prime endothelial cells to modulate subsequent thrombin response

The in vivo endothelial cell translatome is highly heterogeneous across vascular beds

Endothelial cells (ECs) are highly specialized across vascular beds. However, given their interspersed anatomic distribution, comprehensive characterization of the molecular basis for this heterogeneity in vivo has been limited. By applying endothelial-specific translating ribosome affinity purification (EC-TRAP) combined with high-throughput RNA sequencing analysis, we identified pan EC-enriched genes and tissue-specific EC transcripts, which include both established markers and genes previously unappreciated for their presence in ECs. In addition, EC-TRAP limits changes in gene expression after EC isolation and in vitro expansion, as well as rapid vascular bed-specific shifts in EC gene expression profiles as a result of the enzymatic tissue dissociation required to generate single-cell suspensions for fluorescence-activated cell sorting or single-cell RNA sequencing analysis. Comparison of our EC-TRAP with published single-cell RNA sequencing data further demonstrates considerably greater sensitivity of EC-TRAP for the detection of low abundant transcripts. Application of EC-TRAP to examine the in vivo host response to lipopolysaccharide (LPS) revealed the induction of gene expression programs associated with a native defense response, with marked differences across vascular beds. Furthermore, comparative analysis of whole-tissue and TRAP-selected mRNAs identified LPS-induced differences that would not have been detected by whole-tissue analysis alone. Together, these data provide a resource for the analysis of EC-specific gene expression programs across heterogeneous vascular beds under both physiologic and pathologic conditions

Single platelet and megakaryocyte morpho-dynamics uncovered by multicolor reporter mouse strains in vitro and in vivo

Visualizing cell behavior and effector function on a single cell level has been crucial for understanding key aspects of mammalian biology. Due to their small size, large number and rapid recruitment into thrombi, there is a lack of data on fate and behavior of individual platelets in thrombosis and hemostasis. Here we report the use of platelet lineage restricted multi-color reporter mouse strains to delineate platelet function on a single cell level. We show that genetic labeling allows for single platelet and megakaryocyte tracking and morphological analysis in vivo and in vitro, while not affecting lineage functions. Using Credriven Confetti expression, we provide insights into temporal gene expression patterns as well as spatial clustering of megakaryocytes in the bone marrow. In the vasculature, shape analysis of activated platelets recruited to thrombi identifies ubiquitous filopodia formation with no evidence of lamellipodia formation. Single cell tracking in complex thrombi reveals prominent myosin-dependent motility of platelets and highlights thrombus formation as a highly dynamic process amenable to modification and intervention of the acto-myosin cytoskeleton. Platelet function assays combining flow cytrometry, as well as in vivo, ex vivo and in vitro imaging show unaltered platelet functions of multicolor reporter mice compared to WT controls. In conclusion, platelet lineage multicolor reporter mice prove useful in furthering our understanding of platelet and megakaryocyte biology on a single cell level

Vascular endothelial growth factor is an important determinant of sepsis morbidity and mortality

Sepsis, the systemic inflammatory response to infection, is a leading cause of morbidity and mortality. The mechanisms of sepsis pathophysiology remain obscure but are likely to involve a complex interplay between mediators of the inflammatory and coagulation pathways. An improved understanding of these mechanisms should provide an important foundation for developing novel therapies. In this study, we show that sepsis is associated with a time-dependent increase in circulating levels of vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) in animal and human models of sepsis. Adenovirus-mediated overexpression of soluble Flt-1 (sFlt-1) in a mouse model of endotoxemia attenuated the rise in VEGF and PlGF levels and blocked the effect of endotoxemia on cardiac function, vascular permeability, and mortality. Similarly, in a cecal ligation puncture (CLP) model, adenovirus–sFlt-1 protected against cardiac dysfunction and mortality. When administered in a therapeutic regimen beginning 1 h after the onset of endotoxemia or CLP, sFlt peptide resulted in marked improvement in cardiac physiology and survival. Systemic administration of antibodies against the transmembrane receptor Flk-1 but not Flt-1 protected against sepsis mortality. Adenovirus-mediated overexpression of VEGF but not PlGF exacerbated the lipopolysaccharide-mediated toxic effects. Together, these data support a pathophysiological role for VEGF in mediating the sepsis phenotype

Identifying Patients at High Risk of a Cardiovascular Event in the Near Future Current Status and Future Directions: Report of a National Heart, Lung, and Blood Institute Working Group

The National Heart, Lung, and Blood Institute convened a working group to provide basic and clinical research recommendations to the National Heart, Lung, and Blood Institute on the development of an integrated approach for identifying those individuals who are at high risk for a cardiovascular event such as acute coronary syndromes (ACS) or sudden cardiac death in the “near term.” The working group members defined near-term as occurring within 1 year of the time of assessment. The participants reviewed current clinical cardiology practices for risk assessment and state-of-the-science techniques in several areas, including biomarkers, proteomics, genetics, psychosocial factors, imaging, coagulation, and vascular and myocardial susceptibility. This report presents highlights of these reviews and a summary of suggested research directions