Low
molecular weight heparins (LMWHs) are heterogeneous, polydisperse,
and highly negatively charged mixtures of glycosaminoglycan chains
prescribed as anticoagulants. The detailed characterization of LMWH
is important for the drug quality assurance and for new drug research
and development. In this study, online hydrophilic interaction chromatography
(HILIC) Fourier transform mass spectrometry (FTMS) was applied to
analyze the oligosaccharide fragments of LMWHs generated by heparin
lyase II digestion. More than 40 oligosaccharide fragments of LMWH
were quantified and used to compare LMWHs prepared by three different
manufacturers. The quantified fragment structures included unsaturated
disaccharides/oligosaccharides arising from the prominent repeating
units of these LMWHs, 3-O-sulfo containing tetrasaccharides
arising from their antithrombin III binding sites, 1,6-anhydro ring-containing
oligosaccharides formed during their manufacture, saturated uronic
acid oligosaccharides coming from some chain nonreducing ends, and
oxidized linkage region oligosaccharides coming from some chain reducing
ends. This bottom-up approach provides rich detailed structural analysis
and quantitative information with high accuracy and reproducibility.
When combined with the top-down approach, HILIC LC-FTMS based analysis
should be suitable for the advanced quality control and quality assurance
in LMWH production.
Objective
The role of hemoglobin and myoglobin in the cardiovascular system is well established, yet other globins in this context are poorly characterized. Here, we examined the expression and function of cytoglobin (CYGB) during vascular injury.
Approach and Results
We characterized CYGB content in intact vessels and primary vascular smooth muscle (VSM) cells and used two different vascular injury models to examine the functional significance of CYGB in vivo. We found that CYGB was strongly expressed in medial arterial VSM and human veins. In vitro and in vivo studies indicated that CYGB was lost upon VSM cell de-differentiation. In the rat balloon angioplasty model, site-targeted delivery of adenovirus encoding shRNA specific for CYGB prevented its re-expression and decreased neointima formation. Similarly, four weeks after complete ligation of the left common carotid, Cygb knockout mice displayed little to no evidence of neointimal hyperplasia in contrast to their wild-type littermates. Mechanistic studies in the rat indicated that this was primarily associated with increased medial cell loss, TUNEL staining, and caspase-3 activation, all indicative of prolonged apoptosis. In vitro, CYGB could be re-expressed upon VSM stimulation with cytokines and hypoxia and loss of CYGB sensitized human and rat aortic VSM cells to apoptosis. This was reversed upon antioxidant treatment or NOS2 inhibition.
Conclusions
These results indicate that CYGB is expressed in vessels primarily in differentiated medial vascular smooth muscle cells where it regulates neointima formation and inhibits apoptosis after injury.
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