Simulation of Platelet, Thrombus and Erythrocyte Hydrodynamic Interactions in a 3D Arteriole with In Vivo Comparison

Cylindrical blood vessels, ellipsoid platelets and biconcave-shaped deformable erythrocytes (RBCs) are important participants in hemostasis and thrombosis. However, due to the challenge of combining these components in simulation tools, few simulation studies have included all of them in realistic three-dimensional models. In the present study, we apply a recently developed simulation model to incorporate these components and analyze the flow in a thrombotic tubular arteriole, particularly the detailed hydrodynamic interactions between the thrombus shape, RBCs and platelets. It was found that at certain azimuth positions, the velocity drops in the proximity of both the upstream and downstream edge of the thrombus, which is accompanied by a rapid velocity increase in the narrowed region. The RBCs alter the flow profiles significantly from the typical low Reynolds (Re) number flow, and also enhance the deposition of free flowing platelets onto the thrombus. By evaluating the platelet-thrombus interaction and platelet-RBC interaction together, several mechanisms of platelet deposition augmentation are identified. With in vivo data comparison, our model illustrates the potential of future thrombosis studies that incorporate detailed receptor-ligand adhesion modules

Adhesive mechanisms governing interferon-producing cell recruitment into lymph nodes

Natural interferon-producing cells (IPCs) are found in peripheral lymph nodes (PLNs), where they support NK cell, T cell, and B cell responses to pathogens. However, their route of entry and the adhesive mechanisms used to gain access to PLNs remain poorly defined. We report that IPCs can enter PLNs via a hematogenous route, which involves a multistep adhesive process, and that transmigration is enhanced by inflammation. Results indicate that L-selectin on IPCs is required for efficient attachment and rolling on high endothelial venules in vivo in both nonstimulated and inflamed PLNs. IPCs, however, also possess functional ligands for E-selectin that contribute to this process only in the latter case. In conjunction with selectin-mediated adhesion, both β1- and β2-integrins participate in IPC attachment to the inflamed vessel wall, whereas chemotaxis relies in part on the chemokine receptor CCR5. Identification of the adhesive machinery required for IPC trafficking into PLNs may provide opportunities to regulate immune responses reliant on the activity of these cells

A combination of three distinct trafficking signals mediates axonal targeting and presynaptic clustering of GAD65

The signals involved in axonal trafficking and presynaptic clustering are poorly defined. Here we show that targeting of the γ-aminobutyric acid–synthesizing enzyme glutamate decarboxylase 65 (GAD65) to presynaptic clusters is mediated by its palmitoylated 60-aa NH2-terminal domain and that this region can target other soluble proteins and their associated partners to presynaptic termini. A Golgi localization signal in aa 1–23 followed by a membrane anchoring signal upstream of the palmitoylation motif are required for this process and mediate targeting of GAD65 to the cytosolic leaflet of Golgi membranes, an obligatory first step in axonal sorting. Palmitoylation of a third trafficking signal downstream of the membrane anchoring signal is not required for Golgi targeting. However, palmitoylation of cysteines 30 and 45 is critical for post-Golgi trafficking of GAD65 to presynaptic sites and for its relative dendritic exclusion. Reduction of cellular cholesterol levels resulted in the inhibition of presynaptic clustering of palmitoylated GAD65, suggesting that the selective targeting of the protein to presynaptic termini is dependent on sorting to cholesterol-rich membrane microdomains. The palmitoylated NH2-terminal region of GAD65 is the first identified protein region that can target other proteins to presynaptic clusters

Publication Recommendations to Report Laboratory Data of Neonates - a Modified Delphi Approach

Background: Clinical and analytical information on laboratory data of neonates in scientific publications is sparse and incomplete. Furthermore, interpreting neonatal laboratory data can be complex due to their time-dependent and developmental physiology, and paucity of well-established age-appropriate reference ranges for neonates. This study aims to develop publication recommendations to report laboratory data of neonates to enhance the quality of these data in research and clinical care. Methods: A modified Delphi approach was used to develop recommendations in cooperation with the International Neonatal Consortium. A Core Group, including different stakeholders, was responsible for developing the recommendations, in collaboration with a Reflection Group, responsible for providing additional input. Results: The recommendations were classified into three categories: ‘Clinical Characteristics’, ‘Bio-analytical Information’ and ‘Data-analytical Information’. These were each divided into ‘Core Data’ (always to be reported) and ‘Supplemental Considerations’ (to be reported when considered relevant to the study). Conclusion: Our recommendations provide guidance on standardization of neonatal laboratory data in publications. This will enhance the comparison, replication, and application of study results in research initiatives and clinical practice. Furthermore, these recommendations also serve as foundational work to develop reference ranges for neonatal laboratory values by standardizing the quality of information needed for such efforts. Impact: Standardized reporting of neonatal laboratory data in scientific publications will enhance the comparison, replication, and application of study results in research initiatives and clinical practice, as well as improve reporting to regulatory agencies. To integrate multistakeholder perspectives, a modified Delphi approach was used to develop publication recommendations which strengthens the applicability of the recommendations. Implementation of standardization will likely improve the overall quality of neonatal clinical research and neonatal healthcare. In addition, these recommendations are foundational to develop reference ranges for neonatal laboratory values by standardizing the quality of information needed for such efforts.</p

Effects of oxidized low density lipoprotein, lipid mediators and statins on vascular cell interactions

The integrin heterodimer CD11b/CD18 (alpha M beta 2, Mac-1, CR3) expressed on monocytes or polymorphonuclear leukocytes (PMN) is a receptor for iC3b, fibrinogen, heparin, and for intercellular adhesion molecule (ICAM)-1 on endothelium, crucially contributing to vascular cell interactions in inflammation and atherosclerosis. In this report, we summarize our findings on the effects of lipid mediators and lipid-lowering drugs. Exposure of endothelial cells to oxidized low density lipoprotein (oxLDL) induces upregulation of ICAM-1 and increases adhesion of monocytic cells expressing Mac-1. Inhibition experiments show that monocytes use distinct ligands, i.e. ICAM-1 and heparan sulfate proteoglycans for adhesion to oxLDL-treated endothelium. An albumin-transferable oxLDL activity is inhibited by the antioxidant pyrrolidine dithiocarbamate (PDTC), while 8-epi-prostaglandin F2 alpha (8-epi-PGF2 alpha) or lysophosphatidylcholine had no effect, implicating yet unidentified radicals. Sequential adhesive! and signaling events lead to the firm adhesion of rolling PMN on activated and adherent platelets, which may occupy areas of endothelial denudation. Shear resistant arrest of PMN on thrombin-stimulated platelets in flow conditions requires distinct regions of Mac-1, involving its interactions with fibrinogen bound to platelet alpha llb beta 3, and with other platelet ligands. Both arrest and adhesion strengthening under flow are stimulated by platelet-activating factor and leukotriene B4, but not by the chemokine receptor CXCR2. We tested whether Mac-1-dependent monocyte adhesiveness is affected by inhibitors of hydroxy-methylglutaryl-Coenzyme A reductase (statins) which improve morbidity and survival of patients with coronary heart disease. As compared to controls, adhesion of isolated monocytes to endothelium ex vivo was increased in patients with hypercholesterolemia. Treatment with statins decreased total and low density lipoprotein (LDL) cholesterol plasma levels, surface expression of Mac-1, and resulted in a dramatic reduction of Mac,mediated monocyte adhesion to endothelium. The inhibition of monocyte adhesion was reversed by mevalonate but not LDL in vitro,indicating that isoprenoid precursors are crucial for adhesiveness of Mac-1. Such effects may crucially contribute to the clinical benefit of statins, independent of cholesterol-lowering, and may represent a paradigm for novel, anti-inflammatory mechanisms of action by this class of drugs

Histamine deficiency promotes inflammation-associated carcinogenesis through reduced myeloid maturation and accumulation of CD11b \u3csup\u3e+\u3c/sup\u3eLy6G\u3csup\u3e+\u3c/sup\u3e immature myeloid cells

Histidine decarboxylase (HDC), the unique enzyme responsible for histamine generation, is highly expressed in myeloid cells, but its function in these cells is poorly understood. Here we show that Hdc-knockout mice show a high rate of colon and skin carcinogenesis. Using Hdc-EGFP bacterial artificial chromosome (BAC) transgenic mice in which EGFP expression is controlled by the Hdc promoter, we show that Hdc is expressed primarily in CD11b +Ly6G+ immature myeloid cells (IMCs) that are recruited early on in chemical carcinogenesis. Transplant of Hdc-deficient bone marrow to wild-type recipients results in increased CD11b + Ly6G + cell mobilization and reproduces the cancer susceptibility phenotype of Hdc-knockout mice. In addition, Hdc-deficient IMCs promote the growth of tumor allografts, whereas mouse CT26 colon cancer cells downregulate Hdc expression through promoter hypermethylation and inhibit myeloid cell maturation. Exogenous histamine induces the differentiation of IMCs and suppresses their ability to support the growth of tumor allografts. These data indicate key roles for Hdc and histamine in myeloid cell differentiation and CD11b+Ly6G+IMCs in early cancer development. © 2011 Nature America, Inc. All rights reserved

A selective microRNA-based strategy inhibits restenosis while preserving endothelial function.

Drugs currently approved to coat stents used in percutaneous coronary interventions do not discriminate between proliferating vascular smooth muscle cells (VSMCs) and endothelial cells (ECs). This lack of discrimination delays reendothelialization and vascular healing, increasing the risk of late thrombosis following angioplasty. We developed a microRNA-based (miRNA-based) approach to inhibit proliferative VSMCs, thus preventing restenosis, while selectively promoting reendothelialization and preserving EC function. We used an adenoviral (Ad) vector that encodes cyclin-dependent kinase inhibitor p27(Kip1) (p27) with target sequences for EC-specific miR-126-3p at the 3' end (Ad-p27-126TS). Exogenous p27 overexpression was evaluated in vitro and in a rat arterial balloon injury model following transduction with Ad-p27-126TS, Ad-p27 (without miR-126 target sequences), or Ad-GFP (control). In vitro, Ad-p27-126TS protected the ability of ECs to proliferate, migrate, and form netw! orks. At 2 and 4 weeks after injury, Ad-p27-126TS-treated animals exhibited reduced restenosis, complete reendothelialization, reduced hypercoagulability, and restoration of the vasodilatory response to acetylcholine to levels comparable to those in uninjured vessels. By incorporating miR-126-3p target sequences to leverage endogenous EC-specific miR-126, we overexpressed exogenous p27 in VSMCs, while selectively inhibiting p27 overexpression in ECs. Our proof-of-principle study demonstrates the potential of using a miRNA-based strategy as a therapeutic approach to specifically inhibit vascular restenosis while preserving EC function

Human plasma and serum extracellular small RNA reference profiles and their clinical utility

Circulating extracellular RNAs (exRNAs) have the potential to serve as biomarkers for a wide range of medical conditions. However, limitations in existing exRNA isolation methods and a lack of knowledge on parameters affecting exRNA variability in human samples may hinder their successful discovery and clinical implementation. Using combinations of denaturants, reducing agents, proteolysis, and revised organic extraction, we developed an automated, high-throughput approach for recovery of exRNAs and exDNA from the same biofluid sample. We applied this method to characterize exRNAs from 312 plasma and serum samples collected from 13 healthy volunteers at 12 time points over a 2-month period. Small RNA cDNA library sequencing identified nearly twofold increased epithelial-, muscle-, and neuroendocrine-cell–specific miRNAs in females, while fasting and hormonal cycle showed little effect. External standardization helped to detect quantitative differences in erythrocyte and platelet-specific miRNA contributions and in miRNA concentrations between biofluids. It also helped to identify a study participant with a unique exRNA phenotype featuring a miRNA signature of up to 20-fold elevated endocrine-cell–specific miRNAs and twofold elevated total miRNA concentrations stable for over 1 year. Collectively, these results demonstrate an efficient and quantitative method to discern exRNA phenotypes and suggest that plasma and serum RNA profiles are stable over months and can be routinely monitored in long-term clinical studies