Pentti Mäntymaa

Mechanisms of mechanical load-induced atrial natriuretic peptide secretion: role of endothelin, nitric oxide, and angiotensin II.

Abstract There are three members in the natriuretic peptide hormone family, atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP, brain natriuretic peptide), and C-type natriuretic peptide (CNP), that are involved in the regulation of blood pressure and fluid homeostasis. CNP is found principally in the central nervous system and vascular endothelial cells while ANP and BNP are cardiac hormones. ANP is synthesized mainly in the atria of the normal adult heart, while BNP is produced by both the atria and ventricles. The mechanisms controlling ANP release have been the subject of intense research, and are now fairly well understood. The major determinant of ANP secretion is myocyte stretch. Although much less is known about the factors regulating BNP release from the heart, myocyte stretch has also been reported to stimulate BNP release from both atria and ventricles. However, whether wall stretch acts directly or via factors such as endothelin-1, nitric oxide, or angiotensin II liberated in response to distension has not been established. Recent studies show that by stimulating endothelin type A receptors endothelin plays an important physiological role as a mediator of acute-volume load-induced ANP secretion from atrial myocytes in conscious animals. In fact, endogenous paracrine/autocrine factors liberated in response to atrial wall stretch rather than direct stretch appears to be responsible for activation of ANP secretion in response to volume load, as evidenced by almost complete blockade of ANP secretion during combined inhibition of endothelin type A/B and angiotensin II receptors. Furthermore, under certain experimental conditions angiotensin II and nitric oxide may also exert a significant modulatory effect on stretch-activated ANP secretion. The molecular mechanisms by which endothelin-1, angiotensin II, and nitric oxide synergistically regulate stretch-activated ANP release are yet unclear.

Induction of mitochondrial manganese superoxide dismutase confers resistance to apoptosis in acute myeloblastic leukaemia cells exposed to etoposide

We investigated the possible roles of mitochondrial manganese superoxide dismutase (MnSOD) and bcl‐2 in etoposide‐induced cell death in acute myeloblastic leukaemia (AML) using two subclones of the OCI/AML‐2 cell line, the etoposide‐sensitive (ES) and the etoposide‐resistant (ER), as models. Cell death after 24 h exposure to 10 μmol/l etoposide was about 60% and 70% in the ES subclone and about 20% and 25% in the ER subclone, when analysed by trypan blue and annexin V respectively. Cytochrome c efflux from mitochondria to cytosol was observed after 4 h of exposure in both subclones, whereas the activation of caspase‐3 was not detectable until after 12 h of exposure in the ES subclone and 24 h of exposure in the ER subclone, using Western blotting. The decrease in mitochondrial membrane potential, when analysed by the JC‐1 probe fluorocytometrically, also appeared to take place later in the ER than in the ES subclone. Both subclones showed evident basal expression of MnSOD and bcl‐2 by Western blotting. Etoposide caused a potent induction of MnSOD, more than 400% at 12 h, in the ER but not in the ES subclone. No significant change in bcl‐2 expression could be observed in either of the subclones during exposure to etoposide when analysed by Western blotting or flow cytometry. In conclusion, we suggest that MnSOD might have a special role in the protection of AML cells against etoposide‐induced cell death. Although unable to influence the cytochrome c efflux to cytosol, MnSOD might prevent the disruption of mitochondrial membrane potential, which evidently leads to cell death by releasing various activators of apoptosis.

Efficacy of pre-emptively used plerixafor in patients mobilizing poorly after chemomobilization: a single centre experience

A significant proportion of patients with lymphoid malignancies are hard‐to‐mobilize with a combination of chemotherapy plus granulocyte colony‐stimulating factor (G‐CSF) (chemomobilization). Plerixafor is a novel drug used to improve mobilization of blood stem cells. However, it has been studied mainly in association with G‐CSF mobilization. We evaluated the efficacy of ‘pre‐emptive’ use of plerixafor after chemomobilization in patients who seem to mobilize poorly. During a 15 month period, altogether 63 patients with lymphoid malignancies were admitted to our department for blood stem cell collection. Sixteen patients (25%) received plerixafor after the first mobilization due to the low blood (B) CD34+ cell counts (n = 12) or poor yield of the first collection (n = 4). The median number of plerixafor injections was 1 (1–3). The median B‐CD34+ count after the first plerixafor dose was 39 × 106/L (<1–81) with the median increase of fivefold. Stem cell aphaereses were performed in 14/16 patients (88%) receiving plerixafor and a median of 2.9 × 106/kg (1.6–6.1) CD34+ cells were collected with a median of one aphaeresis (1–3). Altogether 13/16 patients mobilized with a combination of chemomobilization and plerixafor received high‐dose therapy with stem cell support and all engrafted. Pre‐emptive use of plerixafor after chemomobilization is efficient and safe and should be considered in poor mobilizers to avoid collection failure. In patients with low but rising B‐CD34+ counts, the use of plerixafor might be delayed as late mobilization may occur. Further studies are needed to optimize patient selection and timing of plerixafor.

An association between mitochondrial function and all-trans retinoic acid-induced apoptosis in acute myeloblastic leukaemia cells

The present study investigated whether all‐trans retinoic acid (ATRA)‐induced apoptosis in acute myeloblastic leukaemia (AML) is related to changes in mitochondrial function. Two human AML cell lines, OU‐AML‐3 and OU‐AML‐7, known to be inducible to time‐dependent apoptosis of varying degrees by ATRA, were used. Apoptosis induced by ATRA was shown to be a slow event. It was detected by the DNA electrophoretic method and cytofluorimetrical annexin V assay after 48 h exposure, and by morphology and polyADPribose polymerase (PARP) cleavage after 72 h exposure of AML cells to ATRA. The efflux of mitochondrial cytochrome c to cytosol was notable in Western blotting after 48 h exposure of the cells to ATRA and was observed before the drop in the mitochondrial membrane potential, which only took place after 72 h exposure, when measured by flow cytometry and a JC‐1 probe. The apoptotic events in mitochondria were more evident in the OU‐AML‐3 than the OU‐AML‐7 cell line. This might relate to the different bcl‐2 contents of the cell lines: the basic bcl‐2 levels of the OU‐AML‐7 cell line were almost twofold compared to that of the OU‐AML‐3 cell line, as analysed by the ELISA method. However, both of the cell lines showed progressive down‐regulation of bcl‐2, which began after 12–24 h exposure of the cells to ATRA as determined by ELISA, Western blotting and flow cytometry. The present results show that mitochondria have a role in ATRA‐induced apoptosis in AML cells and down‐regulation of bcl‐2 is related to it. In view of the previously published studies, the present results underline the fact that the timing of apoptotic events, such as fragmentation of DNA, externalization of phosphatidylserine, cytochrome c efflux, change in mitochondrial membrane potential and cleavage of PARP, are, to a notable extent, cell type and inducer‐dependent.

Blood graft composition after plerixafor injection in patients with NHL

Plerixafor is used to mobilize CD34+ hematopoietic stem cells from bone marrow to circulation. Limited data are available in regard to graft cellular content collected after plerixafor.

Staurosporine, a protein kinase C inhibitor, inhibits atrial natriuretic peptide secretion induced by sarafotoxin, endothelin and phorbol ester

We studied the effects of two peptides of the endothelin/sarafotoxin family, sarafotoxin-b (SRTX-b) and endothelin (ET-1), as well as the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on immunoreactive atrial natriuretic peptide (IR-ANP) release and on haemodynamic parameters (perfusion pressure, heart rate and contractile force) in isolated perfused rat hearts in order to examine the role of intracellular signals in the regulation of ANP secretion. Infusion of SRTX-b at doses of 0.9 and 2.7 nM for 30 min caused a gradual, dose-dependent increase in IR-ANP release and a more rapid coronary vasoconstriction similar to the infusions of ET-1 (2.7 nM) or TPA (46 nM), known to activate protein kinase C in heart cells. A transient inotropic response with a later decrease in contractile force was observed after infusion of each agent. SRTX-b and TPA produced a sustained chronotropic effect, while ET-1 did not significantly affect the heart rate. Infusion of 100 nM of staurosporine, a potent inhibitor of protein kinase C, did not affect basal IR-ANP release into the perfusion fluid but slightly decreased perfusion pressure, heart rate and contractile force. When infused together with SRTX-b, ET-1 or TPA, staurosporine significantly inhibited the ANP secretion, coronary vasoconstriction and changes in cardiac function induced by the peptides or phorbol ester. This study shows that SRTX-b stimulates ANP release with a potency similar to that of ET-1 and that the kinetics of their effects on ANP secretion resemble those of TPA.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood graft lymphocyte subsets after plerixafor injection in non-Hodgkin's lymphoma patients mobilizing poorly with chemotherapy plus granulocyte–colony-stimulating factor

BACKGROUND: A combination of chemotherapy plus granulocyte–colony‐stimulating factor (G‐CSF) (chemomobilization) is commonly used to mobilize CD34+ cells to circulation. Plerixafor, a chemokine CXCR4 antagonist, increases the mobilization of CD34+ cells and may also have effect on graft composition.

CD34+ cell subclasses and lymphocyte subsets in blood grafts collected after various mobilization methods in myeloma patients

BACKGROUND: Cyclophosphamide (CY) combined with granulocyte–colony‐stimulating factor (G‐CSF) is commonly used to mobilize stem cells in multiple myeloma (MM). Plerixafor may also be used with G‐CSF in patients who mobilize poorly or it may be added to chemomobilization to boost mobilization. Limited data are available on graft content collected after various mobilization methods.

Kinetics of blood CD34+ cells after chemotherapy plus G-CSF in poor mobilizers: Implications for pre-emptive plerixafor use

Mobilization and collection of stem cells is difficult in a proportion of patients intended for autologous stem cell transplantation (ASCT). We have evaluated mobilization kinetics of blood CD34+ cells (B-CD34+) to form basis for algorithm to facilitate rational pre-emptive plerixafor use. Altogether 390 chemomobilized patients were included. Forty-three patients (11%) did not reach B-CD34+ count ≥10 × 106/l. Mobilization kinetics differed according to the mobilization capacity observed. Among those who were very poor or inadequate mobilizers (peak B-CD34+ count ≤5 × 106/l and 6–10 × 106/l, respectively), B-CD34+ counts rarely rose after white blood cells (WBC) >5–10 × 109/l, whereas in many standard mobilizers a later rise in CD34+ counts could be observed. Four algorithms based on WBC and CD34+ counts were constructed. According to this patient series, algorithm II (WBC >5 × 109/l and B-CD34+ ≤10 × 106/l) and algorithm IV (WBC >10 × 109/l and B-CD34+ ≤10 × 109/l) were the most applicable. For algorithm II the sensitivity was 0.97 and specificity 1.00, respectively, to identify patients for plerixafor use provided that all patients with B-CD34+ maximum ≤10 × 106/l would have needed plerixafor. This simple model needs a prospective validation.

Quality control of flow cytometry data analysis for evaluation of minimal residual disease in bone marrow from acute leukemia patients during treatment.

Low levels of leukemia cells in the bone marrow, minimal residual disease (MRD), are considered to be a powerful indicator of treatment response in acute lymphatic leukemia (ALL). A Nordic quality assurance program, aimed on standardization of the flow cytometry MRD analysis, has been established before implementation of MRD at cutoff level 10−3 as one of stratifying parameters in next Nordic Society of Pediatric Hematology and Oncology (NOPHO) treatment program for ALL. In 4 quality control (QC) rounds 15 laboratories determined the MRD levels in 48 follow-up samples from 12 ALL patients treated according to NOPHO 2000. Analysis procedures were standardized. For each QC round a compact disc containing data in list-mode files was sent out and results were submitted to a central laboratory. At cutoff level 10−3, which will be applied for clinical decisions, laboratories obtained a high concordance (91.6%). If cutoff level 10−4 was applied, the concordance would be lower (85.3%). The continuing standardization resulted in better concordance in QC3 and QC4 compared with QC1 and QC2. The concordance was higher in precursor B as compared with T-cell ALL. We conclude that after standardization, flow cytometry MRD detection can be reliably applied in international, multicenter treatment protocols.