Fibrocytes mediate intimal hyperplasia post-vascular injury and are regulated by two tissue factor-dependent mechanisms.

BACKGROUND: CD34(+) α-smooth muscle actin (SMA)(+) cells mediate intimal hyperplasia (IH) after mechanical endoluminal injury. We previously found that IH is tissue factor (TF) dependent. The precise phenotype of the CD34(+) cells mediating IH is unknown and the mechanisms of TF are also unknown. OBJECTIVE: To define the phenotype of cells mediating IH and compare the effects of inhibiting TF on different subsets of CD34(+) cells. METHODS: Endoluminal injury was induced in C57BL/6 and two strains of mice expressing a human tissue factor pathway inhibitor (hTFPI) fusion protein on different subsets of CD34(+) cells. Confocal microscopy, immunocytofluorescence and real-time PCR were used to determine phenotype. RESULTS: Neointimal cells in C57BL/6 mice were defined as a subset of fibrocytes (CD34(+) CD45(+) collagen-1(+) ) expressing SMA, CD31, TIE-2, CXCR4 and CXCL12. Similar cells circulated post-injury and were also found in mice expressing hTFPI on CD34(+) CD31(+) cells, though in these mice, hTFPI inhibited CD31(+) fibrocyte hyperplasia, so no IH developed. Mice with hTFPI on all CD34(+) α-SMA(+) cells repaired arteries back to a pre-injured state. No CD31(+) fibrocytes were found in these mice unless an anti-hTFPI antibody was administered. Similar findings in protease activated receptor (PAR)-1-deficient mice suggested hTFPI prevented thrombin signaling through PAR-1. In vitro, thrombin increased the number of CD31(+) fibrocytes. CONCLUSIONS: Inhibition of TF on CD31(+) fibrocytes inhibits IH whereas inhibition on all CD34(+) α-SMA(+) cells (or PAR-1 deficiency) inhibits the appearance of CD31(+) fibrocytes and promotes repair. These data enhance our understanding of IH and suggest novel ways to promote regenerative repair.

Regulation of the cardiomyocyte transcriptome vs translatome by endothelin-1 and insulin: translational regulation of 5' terminal oligopyrimidine tract (TOP) mRNAs by insulin.

BACKGROUND: Changes in cellular phenotype result from underlying changes in mRNA transcription and translation. Endothelin-1 stimulates cardiomyocyte hypertrophy with associated changes in mRNA/protein expression and an increase in the rate of protein synthesis. Insulin also increases the rate of translation but does not promote overt cardiomyocyte hypertrophy. One mechanism of translational regulation is through 5' terminal oligopyrimidine tracts (TOPs) that, in response to growth stimuli, promote mRNA recruitment to polysomes for increased translation. TOP mRNAs include those encoding ribosomal proteins, but the full panoply remains to be established. Here, we used microarrays to compare the effects of endothelin-1 and insulin on the global transcriptome of neonatal rat cardiomyocytes, and on mRNA recruitment to polysomes (i.e. the translatome). RESULTS: Globally, endothelin-1 and insulin (1 h) promoted >1.5-fold significant (false discovery rate < 0.05) changes in expression of 341 and 38 RNAs, respectively. For these transcripts with this level of change there was little evidence of translational regulation. However, 1336 and 712 RNAs had >1.25-fold significant changes in expression in total and/or polysomal RNA induced by endothelin-1 or insulin, respectively, of which approximately 35% of endothelin-1-responsive and approximately 56% of insulin-responsive transcripts were translationally regulated. Of mRNAs for established proteins recruited to polysomes in response to insulin, 49 were known TOP mRNAs with a further 15 probable/possible TOP mRNAs, but 49 had no identifiable TOP sequences or other consistent features in the 5' untranslated region. CONCLUSIONS: Endothelin-1, rather than insulin, substantially affects global transcript expression to promote cardiomyocyte hypertrophy. Effects on RNA recruitment to polysomes are subtle, with differential effects of endothelin-1 and insulin on specific transcripts. Furthermore, although insulin promotes recruitment of TOP mRNAs to cardiomyocyte polysomes, not all recruited mRNAs are TOP mRNAs.

Signaling alterations in activation-induced nonresponsive CD8 T cells.

Costimulation-dependent production and autocrine use of IL-2 by activated CD8 T cells results in initial clonal expansion, but this is transient. The cells quickly become anergic, unable to produce IL-2 in response to Ag and costimulation, irrespective of the form of costimulation. This activation-induced non-responsiveness (AINR) differs from "classical" anergy in that it results despite the cells receiving both signal 1 and signal 2. AINR cells can still proliferate in response to exogenous IL-2, but can no longer produce it. Other TCR-mediated events including cytolytic function and IFN-gamma production are not affected in the AINR state. To characterize the mechanism(s) responsible for lack of IL-2 production in CD8 T cells in the AINR state, microspheres bearing immobilized anti-TCR Abs or peptide-MHC complexes, B7-1, and ICAM-1 were used to provide well-defined stimuli to the cells. Comparison of normal and AINR cells revealed that in AINR cells extracellular signal-regulated kinase (ERK) is upregulated more transiently, Janus kinase activation is substantially reduced, and activation of p38 is eliminated. PMA and ionomycin restored proliferation and IL-2 production in AINR cells, indicating a signaling defect upstream of Ras and protein kinase C. Inhibitors of ERK (PD98059) and of p38 kinase (SB202190) blocked IL-2 mRNA expression and proliferation of both peptide-MHC/B7-1/ICAM-1-stimulated normal cells and PMA/ionomycin-stimulated AINR cells. Together these results demonstrate that activation of at least ERK and p38 is essential for IL-2 production by CD8 T cells and that up-regulation of these mitogen-activated protein kinases, along with Janus kinase, is defective in AINR cells.

Activation of antigen-specific T cells by artificial cell constructs having immobilized multimeric peptide-class I complexes and recombinant B7-Fc proteins.

T cell activation results from the engagement of multiple receptors on T cells by their respective ligands on antigen presenting cells. Studies using artificial cell surface constructs have demonstrated that effective T cell response requires that antigen be presented on a solid surface with dimensions that approximate those of an intact cell. In this report, we describe the cloning and expression of recombinant B7-1-Fc and B7-2-Fc proteins and their incorporation onto 5-microm latex microspheres along with renatured peptide-MHC. These microspheres provide a simple and effective method for the in vitro or in vivo stimulation of antigen-specific T cells under precisely controlled antigen and costimulation conditions.

Differential scanning calorimetric study of the interaction of cholesterol with the major lipids of the Acholeplasma laidlawii B membrane.

It has been proposed that the lower levels of exogenous cholesterol incorporation into the membranes of the sterol-non-requiring as compared to the sterol-requiring mycoplasmas may be due to the much higher glycolipid content of the former and to the reduced ability of glycolipids, as opposed to phospholipids, to incorporate sterols [Efrati et al. (1986) Arch. Biochem. Biophys. 248, 282-288]. In order to test this hypothesis, we have investigated the interaction of cholesterol with the major membrane glyco- and phospholipids of the sterol-non-requiring mycoplasma Acholeplasma laidlawii B, utilizing elaidic acid-homogenous membranes in order to obviate any differences in the nature of cholesterol-lipid interactions due to variations in the fatty acid composition of the different membrane components. Specifically, we have studied the effect of increasing quantities of cholesterol on the thermotropic phase behavior of aqueous dispersions of phosphatidylglycerol, diglucosyl diacylglycerol, and monoglucosyl diacylglycerol, as well as the total membrane polar lipids of this organism, using high-sensitivity differential scanning calorimetry. We find that cholesterol is highly miscible in both the lamellar gel and liquid-crystalline states of phosphatidylglycerol but exhibits limited miscibility in the two neutral glycolipids, particularly in their lamellar gel and crystalline states. We also demonstrate that cholesterol has a limited miscibility in both the lamellar gel and liquid-crystalline states of bilayers composed of the total A. laidlawii B membrane polar lipids. These results demonstrate that the nature of cholesterol-lipid interactions depends markedly on the structure of the glycerolipid polar headgroup and suggests that the incorporation of lower levels of cholesterol into the membranes of the sterol-non-requiring mycoplasmas may indeed be due, at least in part, to their high glycolipid contents. We also show that cholesterol stabilizes the lamellar liquid-crystalline phase of the monoglucosyl diacylglycerol relative to the inverted hexagonal phase at all sterol concentrations, in contrast to the effects of cholesterol on dielaidoylphosphatidylethanolamine, which destabilizes the lamellar liquid-crystalline phase at low concentrations.