Atherosusceptible Shear Stress Activates Endoplasmic Reticulum Stress to Promote Endothelial Inflammation.

Atherosclerosis impacts arteries where disturbed blood flow renders the endothelium susceptible to inflammation. Cytokine activation of endothelial cells (EC) upregulates VCAM-1 receptors that target monocyte recruitment to atherosusceptible regions. Endoplasmic reticulum (ER) stress elicits EC dysregulation in metabolic syndrome. We hypothesized that ER plays a central role in mechanosensing of atherosusceptible shear stress (SS) by signaling enhanced inflammation. Aortic EC were stimulated with low-dose TNFα (0.3 ng/ml) in a microfluidic channel that produced a linear SS gradient over a 20mm field ranging from 0-16 dynes/cm2. High-resolution imaging of immunofluorescence along the monolayer provided a continuous spatial metric of EC orientation, markers of ER stress, VCAM-1 and ICAM-1 expression, and monocyte recruitment. VCAM-1 peaked at 2 dynes/cm2 and decreased to below static TNFα-stimulated levels at atheroprotective-SS of 12 dynes/cm2, whereas ICAM-1 rose to a maximum in parallel with SS. ER expansion and activation of the unfolded protein response also peaked at 2 dynes/cm2, where IRF-1-regulated VCAM-1 expression and monocyte recruitment also rose to a maximum. Silencing of PECAM-1 or key ER stress genes abrogated SS regulation of VCAM-1 transcription and monocyte recruitment. We report a novel role for ER stress in mechanoregulation at arterial regions of atherosusceptible-SS inflamed by low-dose TNFα

Oxylipins in triglyceride-rich lipoproteins of dyslipidemic subjects promote endothelial inflammation following a high fat meal.

Elevated triglyceride-rich lipoproteins (TGRL) in circulation is a risk factor for atherosclerosis. TGRL from subjects consuming a high saturated fat test meal elicited a variable inflammatory response in TNFα-stimulated endothelial cells (EC) that correlated strongly with the polyunsaturated fatty acid (PUFA) content. This study investigates how the relative abundance of oxygenated metabolites of PUFA, oxylipins, is altered in TGRL postprandially, and how these changes promote endothelial inflammation. Human aortic EC were stimulated with TNFα and treated with TGRL, isolated from subjects' plasma at fasting and 3.5 hrs postprandial to a test meal high in saturated fat. Endothelial VCAM-1 surface expression stimulated by TNFα provided a readout for atherogenic inflammation. Concentrations of esterified and non-esterified fatty acids and oxylipins in TGRL were quantified by mass spectrometry. Dyslipidemic subjects produced TGRL that increased endothelial VCAM-1 expression by ≥35%, and exhibited impaired fasting lipogenesis activity and a shift in soluble epoxide hydrolase and lipoxygenase activity. Pro-atherogenic TGRL were enriched in eicosapentaenoic acid metabolites and depleted in esterified C18-PUFA-derived diols. Abundance of these metabolites was strongly predictive of VCAM-1 expression. We conclude the altered metabolism in dyslipidemic subjects produces TGRL with a unique oxylipin signature that promotes a pro-atherogenic endothelial phenotype

The role of atrial natriuretic peptide to attenuate inflammation in a mouse skin wound and individually perfused rat mesenteric microvessels.

We tested the hypothesis that the anti-inflammatory actions of atrial natriuretic peptide (ANP) result from the modulation of leukocyte adhesion to inflamed endothelium and not solely ANP ligation of endothelial receptors to stabilize endothelial barrier function. We measured vascular permeability to albumin and accumulation of fluorescent neutrophils in a full-thickness skin wound on the flank of LysM-EGFP mice 24 h after formation. Vascular permeability in individually perfused rat mesenteric microvessels was also measured after leukocytes were washed out of the vessel lumen. Thrombin increased albumin permeability and increased the accumulation of neutrophils. The thrombin-induced inflammatory responses were attenuated by pretreating the wound with ANP (30 min). During pretreatment ANP did not lower permeability, but transiently increased baseline albumin permeability concomitant with the reduction in neutrophil accumulation. ANP did not attenuate acute increases in permeability to histamine and bradykinin in individually perfused rat microvessels. The hypothesis that anti-inflammatory actions of ANP depend solely on endothelial responses that stabilize the endothelial barrier is not supported by our results in either individually perfused microvessels in the absence of circulating leukocytes or the more chronic skin wound model. Our results conform to the alternate hypothesis that ANP modulates the interaction of leukocytes with the inflamed microvascular wall of the 24 h wound. Taken together with our previous observations that ANP reduces deformability of neutrophils and their strength of attachment, rolling, and transvascular migration, these observations provide the basis for additional investigations of ANP as an anti-inflammatory agent to modulate leukocyte-endothelial cell interactions

Hematopoietic Stem and Progenitor Cells as Effectors in Innate Immunity

Recent research has shed light on novel functions of hematopoietic stem and progenitor cells (HSPC). While they are critical for maintenance and replenishment of blood cells in the bone marrow, these cells are not limited to the bone marrow compartment and function beyond their role in hematopoiesis. HSPC can leave bone marrow and circulate in peripheral blood and lymph, a process often manipulated therapeutically for the purpose of transplantation. Additionally, these cells preferentially home to extramedullary sites of inflammation where they can differentiate to more mature effector cells. HSPC are susceptible to various pathogens, though they may participate in the innate immune response without being directly infected. They express pattern recognition receptors for detection of endogenous and exogenous danger-associated molecular patterns and respond not only by the formation of daughter cells but can themselves secrete powerful cytokines. This paper summarizes the functional and phenotypic characterization of HSPC, their niche within and outside of the bone marrow, and what is known regarding their role in the innate immune response

Mechanisms of B cell Synapse Formation Predicted by Stochastic Simulation

The clustering of B cell receptor (BCR) molecules and the formation of the protein segregation structure known as the immunological synapse appears to precede antigen (Ag) uptake by B cells. The mature B cell synapse is characterized by a central cluster of BCR/Ag molecular complexes surrounded by a ring of LFA-1/ICAM-1 complexes. Recent experimental evidence shows receptor clustering in B cells can occur via mechanical or signaling-driven processes. An alternative mechanism of diffusion and affinity-dependent binding has been proposed to explain synapse formation in the absence of signaling-driven processes. In this work, we investigated the biophysical mechanisms that drive immunological synapse formation in B cells across the physiological range of BCR affinity (KA~10^6-10^10 M-1) through computational modeling. Our computational approach is based on stochastic simulation of diffusion and reaction events with a clearly defined mapping between probabilistic parameters of our model and their physical equivalents. We show that a diffusion-and-binding mechanism is sufficient to drive synapse formation only at low BCR affinity and for a relatively stiff B cell membrane that undergoes little deformation. We thus predict the need for alternative mechanisms: a difference in the mechanical properties of BCR/Ag and LFA-1/ICAM-1 bonds and/or signaling driven processes.Comment: 35 pages, 11 figures; Supplemental Materials adde

Catecholamine stress alters neutrophil trafficking and impairs wound healing by β2-adrenergic receptor-mediated upregulation of IL-6.

Stress-induced hormones can alter the inflammatory response to tissue injury; however, the precise mechanism by which epinephrine influences inflammatory response and wound healing is not well defined. Here we demonstrate that epinephrine alters the neutrophil (polymorphonuclear leukocyte (PMN))-dependent inflammatory response to a cutaneous wound. Using noninvasive real-time imaging of genetically tagged PMNs in a murine skin wound, chronic, epinephrine-mediated stress was modeled by sustained delivery of epinephrine. Prolonged systemic exposure of epinephrine resulted in persistent PMN trafficking to the wound site via an IL-6-mediated mechanism, and this in turn impaired wound repair. Further, we demonstrate that β2-adrenergic receptor-dependent activation of proinflammatory macrophages is critical for epinephrine-mediated IL-6 production. This study expands our current understanding of stress hormone-mediated impairment of wound healing and provides an important mechanistic link to explain how epinephrine stress exacerbates inflammation via increased number and lifetime of PMNs

Evolvable Mars Campaign Long Duration Habitation Strategies: Architectural Approaches to Enable Human Exploration Missions

The Evolvable Mars Campaign (EMC) is the current NASA Mars mission planning effort which seeks to establish sustainable, realistic strategies to enable crewed Mars missions in the mid-2030s timeframe. The primary outcome of the Evolvable Mars Campaign is not to produce "The Plan" for sending humans to Mars, but instead its intent is to inform the Human Exploration and Operations Mission Directorate near-term key decisions and investment priorities to prepare for those types of missions. The FY'15 EMC effort focused upon analysis of integrated mission architectures to identify technically appealing transportation strategies, logistics build-up strategies, and vehicle designs for reaching and exploring Mars moons and Mars surface. As part of the development of this campaign, long duration habitats are required which are capable of supporting crew with limited resupply and crew abort during the Mars transit, Mars moons, and Mars surface segments of EMC missions. In particular, the EMC design team sought to design a single, affordable habitation system whose manufactured units could be outfitted uniquely for each of these missions and reused for multiple crewed missions. This habitat system must provide all of the functionality to safely support 4 crew for long durations while meeting mass and volume constraints for each of the mission segments set by the chosen transportation architecture and propulsion technologies. This paper describes several proposed long-duration habitation strategies to enable the Evolvable Mars Campaign through improvements in mass, cost, and reusability, and presents results of analysis to compare the options and identify promising solutions. The concepts investigated include several monolithic concepts: monolithic clean sheet designs, and concepts which leverage the co-manifested payload capability of NASA's Space Launch System (SLS) to deliver habitable elements within the Universal Payload Adaptor between the SLS upper stage and the Orion/Service module on the top of the vehicle. Multiple modular habitat options for Mars surface and in-space missions are also considered with various functionality and volume splits between modules to find the best balance of reducing the single largest mass which must be delivered to a destination and reducing the number of separate elements which must be launched. Analysis results presented for each of these concepts in this paper include mass/volume/power sizing using parametric sizing tools, identification of unique operational constraints, and limited comments on the additional impacts of reusability/dormancy on system design. Finally, recommendations will be made for promising solutions which will be carried forward for consideration in the Evolvable Mars Campaign work

Neutrophil Mechanosignaling Promotes Integrin Engagement With Endothelial Cells and Motility Within Inflamed Vessels

Neutrophils are the most motile of mammalian cells, a feature that enables them to protect the host against the rapid spread of pathogens from tissue into the circulatory system. A critical process is the recruitment of neutrophils to inflamed endothelium within post-capillary venules. This occurs through cooperation between at least four families of adhesion molecules and G-protein coupled signaling receptors. These adhesion molecules convert the drag force induced by blood flow acting on the cell surface into bond tension that resists detachment. A common feature of selectin-glycoprotein tethering and integrin-ICAM bond formation is the mechanics by which force acting on these specific receptor-ligand pairs influences their longevity, strength, and topographic organization on the plasma membrane. Another distinctly mechanical aspect of neutrophil guidance is the capacity of adhesive bonds to convert external mechanical force into internal biochemical signals through the transmission of force from the outside-in at focal sites of adhesive traction on inflamed endothelium. Within this region of the plasma membrane, we denote the inflammatory synapse, Ca2+ release, and intracellular signaling provide directional cues that guide actin assembly and myosin driven motive force. This review provides an overview of how bond formation and outside-in signaling controls neutrophil recruitment and migration relative to the hydrodynamic shear force of blood flow