Sample Preparation and Analysis for Imaging Mass Spectrometry.

Imaging mass spectrometry (IMS) is a novel quantitative technique used to investigative diverse biomolecules in tissue sections. Specifically, IMS uses analytical separation of mass spectrometry to determine the spatial distribution of certain lipids and/or proteins located directly on biological sections from a single tissue sample. Typically, IMS is combined with histological analysis to reveal additional distribution details of characterized biomolecules including cell type and/or subcellular localization. In this chapter, we describe the use of Matrix-Assisted Laser Desorption/Ionization (MALDI) Time-Of-Flight/Time-Of-Flight (TOF/TOF) to analyze various cholesterol and phosphatidylcholine species in atherosclerotic plaque of murine heart aortic valves. In particular, we detail animals used, tissue collection, preparation, matrix application, spectra acquisition for generating a color-coded image based on IMS spectral characteristics.

Oxidative stress induces early-onset apoptosis of vascular smooth muscle cells and neointima formation in response to injury.

The present study dissects the mechanisms underlying the rapid onset of apoptosis that precedes post injury vascular remodelling. Using the rat balloon injury model, we demonstrated that a significant number of arterial vascular smooth muscle cells (VSMC) undergo apoptosis at 90 min after the procedure. This apoptotic wave caused significant loss in media cellularity (>90%) over the next 3 h and was accompanied by a marked accumulation of oxidative stress by-products in the vascular wall. Early apoptotic VSMC were rich in p38 mitogen-activated protein kinase (MAPK) and the transcription factor c-Jun and secreted IL-6 and GRO/KC into the milieu as determined using multiplex bead assays. Neointima thickness increased steadily starting on day 3 as a result of pronounced repopulation of the media. A second apoptotic wave that was detected at 14 days after injury affected mostly the neointima and was insufficient to control hyperplasia. Suppression of reactive oxygen species (ROS) production using either the NAD(P)H oxidase inhibitor VAS2870 or pegylated superoxide dismutase (PEG-SOD) significantly decreased the number of apoptotic cells during the first apoptotic wave and showed a trend towards reduction in the neointima-to-media thickness ratio at 30 days post injury. These results indicate that oxidative stress in response to injury induces early-onset apoptosis of VSMC through the activation of redox-sensible MAPK pro-apoptotic pathways. This remodelling process leads to the local accumulation of inflammatory cytokines and repopulation of the media, which ultimately contribute to neointima formation.

CD4(+) lymphocytes improve venous blood flow in experimental arteriovenous fistulae.

BACKGROUND: The role of immune cells in arteriovenous fistulae (AVF) maturation is poorly understood and has received, until quite recently, little attention. This study examines the function of T lymphocytes in AVF vascular remodeling. METHODS: Experimental fistulae were created in athymic rnu nude rats lacking mature T lymphocytes and euthymic control animals by anastomosing the left superior epigastric vein to the nearby femoral artery. Blood flow rates, wall morphology, and histologic changes were assessed in AVF 21 days after creation. The effect of CD4(+) lymphocytes on AVF maturation in athymic animals was analyzed by adoptive transfer of cells after fistula creation. RESULTS: The absence of T lymphocytes compromised blood flow in experimental fistulae. Histopathologic inspection of AVF from athymic rats revealed that T-cell immunodeficiency negatively affected venous vascular remodeling, as evidenced by a reduced lumen, a thick muscular layer, and a low number of inflammatory cells compared with control animals. Adoptive transfer of CD4(+) lymphocytes from euthymic rats into athymic animals after fistula creation improved blood flow and reduced intima-media thickness. CONCLUSION: These results point at the protective role of CD4(+) lymphocytes in the remodeling of the AVF vascular wall.

U1 small nuclear RNA variants differentially form ribonucleoprotein particles in vitro.

The U1 small nuclear (sn)RNA participates in splicing of pre-mRNAs by recognizing and binding to 5' splice sites at exon/intron boundaries. U1 snRNAs associate with 5' splice sites in the form of ribonucleoprotein particles (snRNPs) that are comprised of the U1 snRNA and 10 core components, including U1A, U1-70K, U1C and the 'Smith antigen', or Sm, heptamer. The U1 snRNA is highly conserved across a wide range of taxa; however, a number of reports have identified the presence of expressed U1-like snRNAs in multiple species, including humans. While numerous U1-like molecules have been shown to be expressed, it is unclear whether these variant snRNAs have the capacity to form snRNPs and participate in splicing. The purpose of the present study was to further characterize biochemically the ability of previously identified human U1-like variants to form snRNPs and bind to U1 snRNP proteins. A bioinformatics analysis provided support for the existence of multiple expressed variants. In vitro gel shift assays, competition assays, and immunoprecipitations (IPs) revealed that the variants formed high molecular weight assemblies to varying degrees and associated with core U1 snRNP proteins to a lesser extent than the canonical U1 snRNA. Together, these data suggest that the human U1 snRNA variants analyzed here are unable to efficiently bind U1 snRNP proteins. The current work provides additional biochemical insights into the ability of the variants to assemble into snRNPs.

Macrophage-derived IL-18 and increased fibrinogen deposition are age-related inflammatory signatures of vascular remodeling.

Aging has been associated with pathological vascular remodeling and increased neointimal hyperplasia. The understanding of how aging exacerbates this process is fundamental to prevent cardiovascular complications in the elderly. This study proposes a mechanism by which aging sustains leukocyte adhesion, vascular inflammation, and increased neointimal thickness after injury. The effect of aging on vascular remodeling was assessed in the rat balloon injury model using microarray analysis, immunohistochemistry, and LINCOplex assays. The injured arteries in aging rats developed thicker neointimas than those in younger animals, and this significantly correlated with a higher number of tissue macrophages and increased vascular IL-18. Indeed, IL-18 was 23-fold more abundant in the injured vasculature of aged animals compared with young rats, while circulating levels were similar in both groups of animals. The depletion of macrophages in aged rats with clodronate liposomes ameliorated vascular accumulation of IL-18 and significantly decreased neointimal formation. IL-18 was found to inhibit apoptosis of vascular smooth muscle cells (VSMC) and macrophages, thus favoring both the formation and inflammation of the neointima. In addition, injured arteries of aged rats accumulated 18-fold more fibrinogen-γ than those of young animals. Incubation of rat peritoneal macrophages with immobilized IL-18 increased leukocyte adhesion to fibrinogen and suggested a proinflammatory positive feedback loop among macrophages, VSMC, and the deposition of fibrinogen during neointimal hyperplasia. In conclusion, our data reveal that concentration changes in vascular cytokine and fibrinogen following injury in aging rats contribute to local inflammation and postinjury neointima formation.

Genome-based identification of spliceosomal proteins in the silk moth Bombyx mori.

Pre-messenger RNA splicing is a highly conserved eukaryotic cellular function that takes place by way of a large, RNA-protein assembly known as the spliceosome. In the mammalian system, nearly 300 proteins associate with uridine-rich small nuclear (sn)RNAs to form this complex. Some of these splicing factors are ubiquitously present in the spliceosome, whereas others are involved only in the processing of specific transcripts. Several proteomics analyses have delineated the proteins of the spliceosome in several species. In this study, we mine multiple sequence data sets of the silk moth Bombyx mori in an attempt to identify the entire set of known spliceosomal proteins. Five data sets were utilized, including the 3X, 6X, and Build 2.0 genomic contigs as well as the expressed sequence tag and protein libraries. While homologs for 88% of vertebrate splicing factors were delineated in the Bombyx mori genome, there appear to be several spliceosomal polypeptides absent in Bombyx mori and seven additional insect species. This apparent increase in spliceosomal complexity in vertebrates may reflect the tissue-specific and developmental stage-specific alternative pre-mRNA splicing requirements in vertebrates. Phylogenetic analyses of 15 eukaryotic taxa using the core splicing factors suggest that the essential functional units of the pre-mRNA processing machinery have remained highly conserved from yeast to humans. The Sm and LSm proteins are the most conserved, whereas proteins of the U1 small nuclear ribonucleoprotein particle are the most divergent. These data highlight both the differential conservation and relative phylogenetic signals of the essential spliceosomal components throughout evolution.

Spliceosomal immunophilins.

The spliceosome is a dynamic, macromolecular complex, which removes non-protein-coding introns from pre-mRNA to form mature mRNA in a process known as splicing. This ribonucleoprotein assembly is comprised of five uridine-rich small nuclear RNAs (snRNAs) as well as over 300 proteins. In humans, several of the known proteinaceous splicing factors are members of the immunophilin superfamily. Immunophilins are peptidyl-prolyl cis-trans isomerases that catalyze the conversion of proteins from cis to trans at Xaa-Pro bonds. Our review of the data indicates that some members of this protein family are activators of spliceosomal proteins by way of folding and transport.