Michael B. Filla

Indirect induction of suppressor of cytokine signalling-1 in macrophages stimulated with bacterial lipopolysaccharide: partial role of autocrine/paracrine interferon-alpha/beta.

It has previously been reported by us that a brief prior exposure of mouse bone marrow culture-derived macrophages to bacterial lipopolysaccharide (LPS) resulted in a dramatic reduction in their ability to produce NO in response to a subsequent stimulus with either interferon-gamma (IFN-gamma) or IFN-gamma plus LPS. We show here that this brief exposure to LPS results in an impaired response to subsequently added IFN-gamma. A 2--4 h pretreatment with LPS leads to a dramatic reduction in the IFN-gamma-induced DNA-binding of the transcription factor, signal transducer and activator of transcription 1 alpha (STAT1 alpha). This loss in ability to activate STAT1 alpha temporally correlates with the LPS-induced accumulation of mRNA encoding the suppressor of cytokine signalling-1 (SOCS-1). However, LPS does not directly induce the synthesis of SOCS-1. Rather, LPS induces the synthesis of autocrine/paracrine factors that are the true mediators of SOCS-1 induction. IFN-alpha/beta is one of these mediators, but plays only a partial role in the induction of SOCS-1 because neutralization of LPS-induced IFN-alpha/beta production incompletely inhibits the induction of SOCS-1. We show that mouse IFN-beta directly induces the synthesis of SOCS-1, without the need for prior protein synthesis, and does so with faster kinetics than does LPS. Our results are consistent with the non-specific nature of LPS-induced tolerance and provide a mechanistic insight into nonspecificity; LPS indirectly induces the synthesis of a protein mediator, SOCS-1, which inhibits the signalling that is induced by IFN-gamma.

Histamine induces Toll-like receptor 2 and 4 expression in endothelial cells and enhances sensitivity to Gram-positive and Gram-negative bacterial cell wall components

Histamine is a major inflammatory molecule released from the mast cell, and is known to activate endothelial cells. However, its ability to modulate endothelial responses to bacterial products has not been evaluated. In this study we determined the ability of histamine to modulate inflammatory responses of endothelial cells to Gram‐negative and Gram‐positive bacterial cell wall components and assessed the role of Toll‐like receptors (TLR) 2 and 4 in the co‐operation between histamine and bacterial pathogens. Human umbilical vein endothelial cells (HUVEC) were incubated with lipopolysaccharide (LPS), lipoteichoic acid (LTA), or peptidoglycan (PGN) in the presence or absence of histamine, and the expression and release of interleukin‐6 (IL‐6), and NF‐κB translocation were determined. The effect of histamine on the expression of mRNA and proteins for TLR2 and TLR4 was also evaluated. Incubation of HUVEC with LPS, LTA and PGN resulted in marked enhancement of IL‐6 mRNA expression and IL‐6 secretion. Histamine alone markedly enhanced IL‐6 mRNA expression in HUVEC, but it did not stimulate proportional IL‐6 release. When HUVEC were incubated with LPS, LTA, or PGN in the presence of histamine marked amplification of both IL‐6 production and mRNA expression was noted. HUVEC constitutively expressed TLR2 and TLR4 mRNA and proteins, and these were further enhanced by histamine. The expression of mRNAs encoding MD‐2 and MyD88, the accessory molecules associated with TLR signalling, were unchanged by histamine treatment. These results demonstrate that histamine up‐regulates the expression of TLR2 and TLR4 and amplifies endothelial cell inflammatory responses to Gram‐negative and Gram‐positive bacterial components.

Dynamic analysis of vascular morphogenesis using transgenic quail embryos.

Background One of the least understood and most central questions confronting biologists is how initially simple clusters or sheet-like cell collectives can assemble into highly complex three-dimensional functional tissues and organs. Due to the limits of oxygen diffusion, blood vessels are an essential and ubiquitous presence in all amniote tissues and organs. Vasculogenesis, the de novo self-assembly of endothelial cell (EC) precursors into endothelial tubes, is the first step in blood vessel formation [1]. Static imaging and in vitro models are wholly inadequate to capture many aspects of vascular pattern formation in vivo, because vasculogenesis involves dynamic changes of the endothelial cells and of the forming blood vessels, in an embryo that is changing size and shape. Methodology/Principal Findings We have generated Tie1 transgenic quail lines Tg(tie1:H2B-eYFP) that express H2B-eYFP in all of their endothelial cells which permit investigations into early embryonic vascular morphogenesis with unprecedented clarity and insight. By combining the power of molecular genetics with the elegance of dynamic imaging, we follow the precise patterning of endothelial cells in space and time. We show that during vasculogenesis within the vascular plexus, ECs move independently to form the rudiments of blood vessels, all while collectively moving with gastrulating tissues that flow toward the embryo midline. The aortae are a composite of somatic derived ECs forming its dorsal regions and the splanchnic derived ECs forming its ventral region. The ECs in the dorsal regions of the forming aortae exhibit variable mediolateral motions as they move rostrally; those in more ventral regions show significant lateral-to-medial movement as they course rostrally. Conclusions/Significance The present results offer a powerful approach to the major challenge of studying the relative role(s) of the mechanical, molecular, and cellular mechanisms of vascular development. In past studies, the advantages of the molecular genetic tools available in mouse were counterbalanced by the limited experimental accessibility needed for imaging and perturbation studies. Avian embryos provide the needed accessibility, but few genetic resources. The creation of transgenic quail with labeled endothelia builds upon the important roles that avian embryos have played in previous studies of vascular development.

Low responsiveness to IFN-γ, after pretreatment of mouse macrophages with lipopolysaccharides, develops via diverse regulatory pathways

We have investigated the mechanisms by which prior exposure of mouse macrophages to lipopolysaccharides (LPS) induces a state of low responsiveness to subsequent exposure to IFN‐γ. We demonstrate that induction of this state requires both de novo gene expression and the suppression of phosphorylation events that lead to activation of transcription factor Stat1α. These observations are mechanistically consistent with the known induction of suppressors of cytokine signaling (SOCS)‐1 and SOCS‐3 proteins by LPS. In this regard, we demonstrate that overexpression of either SOCS protein suppresses induction of the mouse inducible nitric oxide synthase (iNOS) gene promoter: apparently by suppressing interactions between Stat1α and IFN‐γ activated sites present in both the iNOS, and interferon regulatory factor‐1, gene promoters. The induction of SOCS‐1 and SOCS‐3 by LPS or IFN‐β (an autocrine/paracrine mediator of LPS‐induced SOCS‐1 mRNA synthesis)occurs by way of multiple protein kinase pathways that include protein tyrosine kinases, protein kinase C, and mitogen‐activated protein kinases. These results provide insight that may allow discriminationbetween LPS‐induced inhibition of macrophage functions that are detrimental to the host (e.g. continued exposure to LPS) versus those that might potentially be beneficial (e.g. exposure to subsequent agonists that induce more specific macrophage functions).

Extracellular Matrix Macroassembly Dynamics in Early Vertebrate Embryos

This chapter focuses on the in vivo macroassembly dynamics of fibronectin and fibrillin-2--two prominent extracellular matrix (ECM) components, present in vertebrate embryos at the earliest stages of development. The ECM is an inherently dynamic structure with a well-defined position fate: ECM filaments are not only anchored to and move with established tissue boundaries, but are repositioned prior to the formation of new anatomical features. We distinguish two ECM filament relocation processes-each operating on different length scales. First, ECM filaments are moved by large-scale tissue motion, which rearranges major organ primordia within the embryo. The second type of motion, on the scale of the individual ECM filaments, is driven by local motility and protrusive activity of nearby cells. The motion decomposition is made practically possible by recent advances in microscopy and high-resolution particle image velocimetry algorithms. We demonstrate that both kinds of motion contribute substantially to the establishment of normal ECM structure, and both must be taken into account when attempting to understand ECM macroassembly during embryonic morphogenesis. The tissue-scale motion changes the local amount (density) and the tissue-level structure (e.g., orientation) of ECM fibers. Local reorganization includes filament assembly and the segregation of ECM into specific patterns. Local reorganization takes place most actively at Hensens node and around the primitive streak. These regions are also sites of active cell migration, where fibrillin-2 and fibronectin are often colocalized in ECM globules, and new fibrillin-2 foci are deposited. During filament assembly, the globular patches of ECM are joined into larger linear structures in a hierarchical process: increasingly larger structures are created by the aggregation of smaller units. A future understanding of ECM assembly thus requires the study of the complex interactions between biochemical assembly steps, local cell action, and tissue motion.

Convective tissue movements play a major role in avian endocardial morphogenesis

Endocardial cells play a critical role in cardiac development and function, forming the innermost layer of the early (tubular) heart, separated from the myocardium by extracellular matrix (ECM). However, knowledge is limited regarding the interactions of cardiac progenitors and surrounding ECM during dramatic tissue rearrangements and concomitant cellular repositioning events that underlie endocardial morphogenesis. By analyzing the movements of immunolabeled ECM components (fibronectin, fibrillin-2) and TIE1 positive endocardial progenitors in time-lapse recordings of quail embryonic development, we demonstrate that the transformation of the primary heart field within the anterior lateral plate mesoderm (LPM) into a tubular heart involves the precise co-movement of primordial endocardial cells with the surrounding ECM. Thus, the ECM of the tubular heart contains filaments that were associated with the anterior LPM at earlier developmental stages. Moreover, endocardial cells exhibit surprisingly little directed active motility, that is, sustained directed movements relative to the surrounding ECM microenvironment. These findings point to the importance of large-scale tissue movements that convect cells to the appropriate positions during cardiac organogenesis.

A transgenic quail model that enables dynamic imaging of amniote embryogenesis.

Embryogenesis is the coordinated assembly of tissues during morphogenesis through changes in individual cell behaviors and collective cell movements. Dynamic imaging, combined with quantitative analysis, is ideal for investigating fundamental questions in developmental biology involving cellular differentiation, growth control and morphogenesis. However, a reliable amniote model system that is amenable to the rigors of extended, high-resolution imaging and cell tracking has been lacking. To address this shortcoming, we produced a novel transgenic quail that ubiquitously expresses nuclear localized monomer cherry fluorescent protein (chFP). We characterize the expression pattern of chFP and provide concrete examples of how Tg(PGK1:H2B-chFP) quail can be used to dynamically image and analyze key morphogenetic events during embryonic stages X to 11. Summary: A novel transgenic quail that ubiquitously expresses nuclear localized CherryFP provides insights into the cellular and morphogenetic events of amniote embryogenesis.

Prolonged exposure of mouse macrophages to IFN-β suppresses transcription of the inducible nitric oxide synthase gene: Altered availability of transcription factor Stat1α

Previous studies from our laboratory have shown that prolonged exposure of mouse macrophages to IFN‐β interferes with their subsequent ability to become activated for tumor cell killing. Data reported here show that such inhibition is due to reduced production of NO, resulting from decreased transcription of the gene that encodes inducible NO synthase (iNOS; EC 1.14.13.39). The molecular basis for such suppression was shown to be, at least in part, decreased nuclear accumulation of tyrosine‐phosphorylated Stat1α (pStat1α), and a consequent change in the nuclear ratio of pStat1α to non‐transactivating pStat1β. Reduced phosphorylation was observed despite the fact that time‐course studies revealed greater than normal quantities of both Stat1α and Stat1β proteins in macrophages that had been pre‐exposed to IFN‐β. The decrease in nuclear pStat1α was demonstrated to involve an increase in the rate of turnover of phosphorylated protein. The homodimeric form of pStat1α is essential for the expression of both the iNOS and IFN‐regulatory factor‐1 genes (the product of the latter is necessary for full expression of the iNOS gene). These results have broad implications, because they suggest that limiting the availability of homodimeric pStat1α is a means by which down‐regulation of genes containing promoter‐linked IFN‐γ‐activated sites might be achieved.

Time-lapse microscopy of macrophages during embryonic vascular development.

Background: Macrophages are present before the onset of blood flow, but very little is known about their function in vascular development. We have developed a technique to concurrently label both endothelial cells and macrophages for time‐lapse microscopy using co‐injection of fluorescently conjugated acetylated low‐density lipoprotein (AcLDL) and phagocytic dye PKH26‐PCL. Results: We characterize double‐labeled cells to confirm specific labeling of macrophages. Double‐labeled cells circulate, roll along the endothelium, and extravasate from vessels. Most observed macrophages are integrated into the vessel wall, showing an endothelial‐like morphology. We used transgenic quail that express a fluorescent protein driven by the endothelial‐specific promoter Tie1 in conjugation with the phagocytic dye to analyze these cells. Circulating PKH26‐PCL‐labeled cells are mostly Tie1−, but those which have integrated into the vessel wall are largely Tie1+. The endothelial‐like phagocytic cells were generally stationary during normal vascular development. We, therefore, induced vascular remodeling and found that these cells could be recruited to sites of remodeling. Conclusions: The active interaction of endothelial cells and macrophages support the hypothesis that these cells are involved in vascular remodeling. The presence of phagocytic endothelial‐like cells suggests either a myeloid‐origin to certain endothelial cells or that circulating endothelial cells/hematopoietic stem cells have phagocytic capacity. Developmental Dynamics 241:1423–1431, 2012.

Decellularized Wharton’s Jelly from human umbilical cord as a novel 3D scaffolding material for tissue engineering applications

In tissue engineering, an ideal scaffold attracts and supports cells thus providing them with the necessary mechanical support and architecture as they reconstruct new tissue in vitro and in vivo. This manuscript details a novel matrix derived from decellularized Wharton’s jelly (WJ) obtained from human umbilical cord for use as a scaffold for tissue engineering application. This decellularized Wharton’s jelly matrix (DWJM) contained 0.66 ± 0.12 μg/mg sulfated glycosaminoglycans (GAGs), and was abundant in hyaluronic acid, and completely devoid of cells. Mass spectroscopy revealed the presence of collagen types II, VI and XII, fibronectin-I, and lumican I. When seeded onto DWJM, WJ mesenchymal stem cells (WJMSCs), successfully attached to, and penetrated the porous matrix resulting in a slower rate of cell proliferation. Gene expression analysis of WJ and bone marrow (BM) MSCs cultured on DWJM demonstrated decreased expression of proliferation genes with no clear pattern of differentiation. When this matrix was implanted into a murine calvarial defect model with, green fluorescent protein (GFP) labeled osteocytes, the osteocytes were observed to migrate into the matrix as early as 24 hours. They were also identified in the matrix up to 14 days after transplantation. Together with these findings, we conclude that DWJM can be used as a 3D porous, bioactive and biocompatible scaffold for tissue engineering and regenerative medicine applications.