NPY/Y1 receptor-mediated vasoconstrictory and proliferative effects in pulmonary hypertension.

BACKGROUND AND PURPOSE: Pulmonary arteries (PAs) are innervated, but little is known about the role of neuronal axis in pulmonary hypertension (PH). Here, we have examined the role of the neuropeptide Y (NPY) and its Y1 receptor in PH pathogenesis. EXPERIMENTAL APPROACH: NPY was localized by immunofluorescence. Expression of NPY and Y1 receptor were determined by quantitative PCR. Cellular response to NPY stimulation was assessed by Western blotting, thymidine incorporation and calcium imaging. Wire myography and isolated perfused mouse lung were applied to study pulmonary vasoactive effects of NPY. Selective receptor antagonists were used to assess the contribution of receptor subtypes in mediating NPY effects. KEY RESULTS: Samples from PH patients showed increased NPYergic innervation within the PA wall and higher Y1 receptor expression, compared with donors. However, NPY levels were unchanged in both PA and serum. In the chronic hypoxic mouse model, Y1 receptor were up-regulated, while expression of both NPY and Y1 receptor was increased in the lungs of monocrotaline and SU5416-hypoxia rats. On a functional level, NPY acutely increased intracellular calcium levels and enhanced vasoconstriction of lung vessels preconstricted with adrenaline. Furthermore, NPY stimulated proliferation of human pulmonary arterial smooth muscle cells and activated p38 and PKD pathways. Correspondingly, higher phosphorylation of PKD was observed in remodelled vessels from PH patients. The selective Y1 receptor antagonist, BIBO 3304, concentration-dependently inhibited vasoconstrictive and proliferative effects of NPY. CONCLUSIONS AND IMPLICATIONS: NPY and Y1 receptor are possible mediators of both vasoconstriction and pulmonary vascular remodelling in PH.

Cell cycle protein expression in vascular smooth muscle cells in vitro and in vivo is regulated through phosphatidylinositol 3-kinase and mammalian target of rapamycin.

Cell cycle progression represents a key event in vascular proliferative diseases, one that depends on an increased rate of protein synthesis. An increase in phosphatidylinositol 3-kinase (PI 3-kinase) activity is associated with vascular smooth muscle cell proliferation, and rapamycin, which blocks the activity of the mammalian target of rapamycin, inhibits this proliferation in vitro and in vivo. We hypothesized that these 2 molecules converge on a critical pathway of translational regulation that is essential for successful upregulation of cell cycle-regulatory proteins in activated smooth muscle cells. p70(S6) kinase, a target of PI 3-kinase and the mammalian target of rapamycin, was rapidly activated on growth factor stimulation of quiescent coronary artery smooth muscle cells and after balloon injury of rat carotid arteries. The translational repressor protein 4E-binding protein 1 was similarly hyperphosphorylated under these conditions. These events were associated with increases in the protein levels of cyclin B1, cyclin D1, cyclin E, cyclin-dependent kinase 1, cyclin-dependent kinase 2, proliferating cell nuclear antigen, and p21(Cip1) in vivo and in vitro, whereas inhibition of the PI 3-kinase signaling pathway with either rapamycin or wortmannin blocked the upregulation of these cell cycle proteins, but not mRNA, and arrested the cells in vitro before S phase. In contrast to findings in other cell types, growth factor- or balloon injury-induced downregulation of the cell cycle inhibitor p27(Kip1) was not affected by rapamycin treatment. These data suggest that cell cycle progression in vascular cells in vitro and in vivo depends on the integrity of the PI 3-kinase signaling pathway in allowing posttranscriptional accumulation of cell cycle proteins.

Expression of the nerve growth factor receptor c-TRK in human myeloid leukaemia cells.

Nerve growth factor (NGF) is of major importance for the survival, development and maintenance of peripheral sympathetic and central neuronal tissue. Most of the cellular effects are mediated by binding to their high-affinity receptor c-TRK, a transmembrane receptor tyrosine kinase. C-TRK protein has been detected in neuronal tissue and also in mast cells, monocytes and some haemopoletic progenitor cells. Here we report c-TRK gene expression in myeloid leukaemic cell lines (HEL, K562 and KG-1) and for the first time in the primary leukaemic cells of 44% (n = 59) of patients with acute myelogenous leukaemia (AML). Moreover, in the human promyelocytic cell line HL-60, c-TRK expression was inducible by differentiation induction with tetradecanoyl-phorbol 13-acetate (TPA). In c-TRK gene-expressing cells the transmembrane receptor tyrosine kinase was detectable by Western blotting and by in vitro kinase assay. In the AML group, c-TRK expression was not correlated to the FAB-classified morphology or any other clinical parameter. In all cases tested we could not detect NGF mRNA by means of reverse transcriptase PCR, excluding an autocrine loop involving the TRK/NGF receptor-ligand system in leukaemogenesis. Our results show another example of possible communication between neuronal and haemopoietic tissue. However, we still lack positive evidence of a c-TRK function in haemopoiesis.

Comparison of different antisense oligonucleotides against the BCR/ABL junction in chronic myelogenous leukemia.

The activity of the BCR/ABL hybrid gene is associated with a growth advantage of the chronic myelogenous leukemia (CML) stem cell. Suppression of BCR/ABL hybrid gene expression can be a valuable tool for leukemic cell purging. Antisense oligonucleotides (ODNs) have the capacity to specifically downregulate gene expression. Data reported on the effect they have on BCR/ABL hybrid gene expression are controversial. We present data illustrating that prolongation of ODN half-life by means of chemical or sequence modification has only limited specific growth suppressive effect on BCR/ABL-positive clonogenic cells in vitro. Compared to unmodified phosphodiester ODNs (PO-ODNs) spanning the BCR/ABL junctions, modified ODNs with either a 3'-GC-clamp (GC-ODNs) or ODNs with one 3'-inverted nucleotide (3'-3' ODNs) to prevent 3'-exonuclease degradation, showed significantly prolonged extra- and intracellular half lives and different subcellular distributions in CML cell lines. In clonogenic assays from patients with CML, the modified ODNs were to some extent able to reduce colonies expressing BCR/ABL (GC-ODNs >3'-3'-ODNs >PO-ODNs). This difference did not become significant statistically. We demonstrate a substantially diminished hybridization efficacy of the modified antisense ODNs used, which may serve as a possible explanation for the failure to augment the leukemic cell purging efficacy.

Unmodified phosphodiester antisense oligodeoxynucleotides to the BCR-ABL junction do not suppress Philadelphia-positive clonogenic cells.

The BCR-ABL fusion gene is directly involved in the pathogenesis of chronic myeloid leukemia (CML). Specific inhibition of the BCR-ABL gene expression with antisense oligodeoxynucleotides has been shown to have profound effects on cell growth in vitro. We examined antisense phosphodiester oligonucleotides (16-mers at a concentration of 60 micrograms/ml) spanning the two possible junction sites K28 (b3a2) and L6 (b2a2) in a clonogenic assay. Single colonies from 9 patients with CML and from patients with normal bone marrow were screened for BCR-ABL expression with a new 'single-tube nested PCR' method. There was a marked reduction in colony number in the CML group and a lesser growth inhibition in the control group. The number and percentage of BCR-ABL-positive colonies, however, was not reduced in the CML group. This indicates a nonspecific growth inhibition only.