Local chromatin interactions contribute to expression of the fibrinogen gene cluster.

Essentials The fibrinogen gene cluster is flanked by CCCTC-binding factor (CTCF) interaction sites. Chromatin looping of the fibrinogen cluster was demonstrated by chromosome conformation capture. Deleting a CTCF interaction site alters chromatin looping and halves fibrinogen expression. Looping of the human fibrinogen locus is functionally linked to fibrinogen gene expression. SUMMARY: Background The coordinately regulated genes encoding human fibrinogen are clustered. This evolutionarily conserved configuration provides a possible mechanism for co-regulation whereby regulatory elements influence gene expression locally. The cluster is flanked by CCCTC-binding factor (CTCF) interaction sites that are candidate insulator regions mediating chromatin looping. Objectives To further our understanding of fibrinogen gene regulation, we aimed to investigate whether interactions exist between parts of the fibrinogen locus and how these contacts contribute to fibrinogen expression. Methods We used chromosome conformation capture in cultured cell lines to detect chromatin interactions at the fibrinogen gene cluster. We generated clonal cell lines where two CTCF interaction sites at one end of the locus were deleted using CRISPR-Cas9-mediated genome editing. Fibrinogen expression and protein production were measured using qRT-PCR and ELISA, respectively. Results We detected proximity between the ends of the fibrinogen locus, regardless of whether cells express fibrinogen. An interaction between the FGA promoter and the edge of the locus was more frequent in fibrinogen-expressing cells. Deletion of a CTCF site at one edge of the cluster altered chromatin interactions, reduced steady-state expression of FGB and FGG mRNA, and led to a halving of secreted fibrinogen. These phenotypes were completely restored by reintroduction of the CTCF interaction motif in previously motif-deleted clones. Conclusions Chromatin interactions are important for the coordinated regulation of the human fibrinogen genes. This finding furthers our comprehension of how fibrinogen is produced and identifies a possible source of variability in plasma fibrinogen levels seen in populations.

Retinoids and activation of PKC induce tissue-type plasminogen activator expression and storage in human astrocytes.

BACKGROUND: Emerging data demonstrate important roles for tissue-type plasminogen activator (t-PA) in the central nervous system (CNS). In contrast to endothelial cells, little is known about the regulation of t-PA gene expression and secretion in astrocytes. OBJECTIVES: The aims of the present study were to investigate whether t-PA gene expression is regulated by retinoids and the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) in human astrocytes, and to study whether t-PA is stored and subject to regulated release from these cells, as with endothelial cells. METHODS: Native human astrocytes were treated with RA and/or PMA. mRNA was quantified by real-time RT-PCR and protein secretion determined by ELISA. Intracellular t-PA immunoreactivity in astrocytes was examined by immunocyto- and histochemistry. RESULTS: RA and/or PMA induced a time-dependent increase in t-PA mRNA and protein levels in astrocytes, reaching 10-fold after combined treatment. This was associated with increased amounts of t-PA storage in intracellular granular structures. Both forskolin and histamine induced regulated release of t-PA. The presence of t-PA in reactive astrocytes was confirmed in human brain tissue. CONCLUSIONS: These data show that RA and PKC activation induce a strong up-regulation of t-PA expression in astrocytes, and increased intracellular storage pools. Moreover, a regulated release of t-PA can be induced from these cells. This raises the possibility that astrocytes contribute to the regulation of extracellular t-PA levels in the CNS.

Characterization of endothelial-like cells derived from human mesenchymal stem cells.

BACKGROUND: Blood-derived endothelial progenitor cells (EPC) have been used to treat ischemic disease. However, the number of EPC that can be obtained from adult blood is limited. OBJECTIVE: To characterize endothelial-like cells obtained from human bone marrow and determine their ability to stimulate new blood vessel formation in vivo. METHODS: Mononuclear cells (MNC) were isolated from human bone marrow or umbilical cord blood and cultured in endothelial growth medium (EGM-2). Mesenchymal stem cells (MSC) were isolated from bone marrow and induced to differentiate into endothelial-like cells (MSCE), or adipocytes or osteocytes by growth in EGM-2, adipogenic or osteogenic medium. RESULTS: Cells obtained by culturing bone marrow MNC in EGM-2 formed cord- or tube-like structures when grown on Matrigel(TM) and expressed several endothelial marker proteins. However, cell morphology and the profile of endothelial marker protein expression were different from those of cord blood-derived EPC (cbEPC). Cells with a similar phenotype were obtained by differentiation of MSC into MSCE, which was accompanied by an increase of endothelial marker proteins and a diminished capacity to differentiate into adipocytes. Subcutaneous implantation of MSCE in collagen plugs in non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice resulted in formation of functional blood vessels that had incorporated the MSCE. CONCLUSIONS: Our results show that MSCE and cbEPC are different cell types. The formation of functional blood vessels by MSCE, combined with high yields and a reduced capacity to differentiate into other cell types compared with MSC, makes these cells potentially useful for autologous therapy of ischemic disease.