Thrombopoietin stimulates colony-forming unit-megakaryocyte proliferation and megakaryocyte maturation independently of cytokines that signal through the gp130 receptor subunit

Thrombopoietin (Tpo), the ligand for the c-Mpl receptor, is a major regulator of megakaryopoiesis. Treatment of mice with Tpo raises the platelet count fourfold within a few days. Conversely, c-mpl knock-out mice have platelet counts that are 15% that of normal. The subunit structure of the c-Mpl receptor is not fully understood. Some cytokines that stimulate megakaryopoiesis (IL-6, IL-11, leukemia inhibitory factor, and oncostatin M) bind to receptors that use gp130 as a signal transduction subunit. For these reasons, we determined whether gp130 function was required for Tpo-induced signal transduction. Murine marrow cells were cultured in semi-solid media in the presence of Tpo or IL-3, with or without a neutralizing anti-gp130 monoclonal antibody (RX187) or a soluble form of c-Mpl receptor (soluble Mpl) that blocks Tpo bioactivity, and the numbers of colony-forming unit-megakaryocyte (CFU-Meg) colonies were counted on day 5. Murine marrow cells were also cultured in suspension under serum-free conditions for 5 days, and megakaryocyte DNA content was measured by flow cytometry, as an index of nuclear maturation. The addition of RX187 did not block Tpo-induced CFU-Meg colony growth nor CFU-Meg nuclear maturation in suspension culture. However, IL-3-induced CFU-Meg colony growth and megakaryocyte nuclear maturation decreased in the presence of RX187. Soluble Mpl completely ablated Tpo-induced CFU-Meg growth, and partially blocked IL- 3-stimulated CFU-Meg growth. Thus the effects of Tpo on megakaryopoiesis in vitro do not depend on cytokines that signal through gp130. Furthermore, it is unlikely that gp 130 serves as a beta chain for the c-Mpl receptor, as Tpo signalling is unimpaired in the presence of RX187. In contrast, the effects of IL-3 on CFU-Meg growth are mediated in part through Tpo and through gp130-signalling cytokines.     

The effect of thrombopoietin on the proliferation and differentiation of murine hematopoietic stem cells

In this study, we explored whether thrombopoietin (Tpo) has a direct in vitro effect on the proliferation and differentiation of long-term repopulating hematopoietic stem cells (LTR-HSC). We previously reported a cell separation method that uses the fluorescence-activated cell sorter selection of low Hoescht 33342/low Rhodamine 123 (low Ho/low Rh) fluorescence cell fractions that are highly enriched for LTR-HSC and can reconstitute lethally irradiated recipients with fewer than 20 cells. Low Ho/low Rh cells clone with high proliferative potential in vitro in the presence of stem cell factor (SCF) + interleukin-3 (IL-3) + IL-6 (90% to 100% HPP-CFC). Tpo alone did not induce proliferation of these low Ho/low Rh cells. However, in combination with SCF or IL-3, Tpo had several synergistic effects on cell proliferation. When Tpo was added to single growth factors (either SCF or IL-3 or the combination of both), the time required for the first cell division of low Ho/low Rh cells was significantly shortened and their cloning efficiency increased substantially. Moreover, the subsequent clonal expansion at the early time points of culture was significantly augmented by Tpo. Low Ho/low Rh cells, when assayed in agar directly after sorting, did not form megakaryocyte colonies in any growth condition tested. Several days of culture in the presence of multiple cytokines were required to obtain colony-forming units-megakaryocyte (CFU-Mk). In contrast, more differentiated, low Ho/high Rh cells, previously shown to contain short- term repopulating hematopoietic stem cells (STR-HSC), were able to form megakaryocyte colonies in agar when cultured in Tpo alone directly after sorting. These data establish that Tpo acts directly on primitive hematopoietic stem cells selected using the Ho/Rh method, but this effect is dependent on the presence of pluripotent cytokines. These cells subsequently differentiate into CFU-Mk, which are capable of responding to Tpo alone. Together with the results of previous reports of its effects on erythroid progenitors, these results suggest that the effects of Tpo on hematopoiesis are greater than initially anticipated.     

Static exercise with congestive heart failure and the response to vasodilating drugs

The hemodynamic response to static exercise in 28 patients with congestive heart failure (CHF) was compared with that in 8 control subjects. Static handgrip exercise at 50% of the maximal voluntary contraction was performed to fatigue. In patients with CHF, pulmonary arterial wedge pressure increased from 20 +/- 18 to 31 +/- 10 mm Hg (p less than 0.001) (mean +/- standard deviation) and systemic vascular resistance increased from 1,730 +/- 454 to 2,151 +/- 724 dynes s cm-5 (p less than 0.001). Although cardiac index did not change significantly, stroke volume index and stroke work index decreased from 24 +/- 6 to 20 +/- 6 ml/m2 (p less than 0.001) and 28 +/- 11 to 25 +/- 12 g-m/s2 (p less than 0.05), respectively. In control subjects, pulmonary arterial wedge pressure did not change significantly; cardiac index increased from 3.6 +/- 0.3 to 4.0 +/- 0.4 liters/min/m2 (p less than 0.05) and systemic vascular resistance increased slightly, from 1,011 +/- 186 to 1,106 +/- 180 dynes s cm-5 (p less than 0.05). The effects of arterial dilation with hydralazine on the response to static exercise were assessed in 10 of the patients with CHF. Compared with predrug exercise, cardiac index increased 68% (p less than 0.01), stroke volume index increased 76% (p less than 0.01) and systemic vascular resistance decreased 47% (p less than 0.01) after administration of hydralazine. Thus, static exercise can have adverse effects on cardiac performance in patients with CHF.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibitors of bradykinin-inactivating enzymes decrease myocardial ischemia/reperfusion injury following 3 and 7 days of reperfusion

Inhibitors of bradykinin (BK)-inactivating enzymes protect from myocardial ischemia/reperfusion injury after short periods of reperfusion. However, protection after 2 to 3 h of reperfusion does not mean that myocardium remains viable for an extended time. Therefore, we examined the effects of inhibitors of angiotensin-converting enzyme (ramiprilat), EP24.11 (cFP-F-pAB), and EP24.15 (cFP-AAF-pAB) in a chronic model of myocardial ischemia/reperfusion injury. A left descending coronary artery was occluded for 30 min in anesthetized rabbits. Saline, ramiprilat, or endopeptidase inhibitors were given after 27 min of occlusion. The BK(2) receptor antagonist HOE140 was administered in certain experiments. After ischemia, the occlusion was released, and the animal allowed to recover for 3 or 7 days. Surgery was then repeated, and the heart removed for determination of infarct size. In separate experiments, the heart was removed after 2 h of reperfusion for determination of BK tissue levels. Ramiprilat and endopeptidase inhibitors reduced infarct size at 3 and 7 days. Combining inhibitors further reduced infarct size after 3 days. The protective effect of the endopeptidase inhibitors was blocked by HOE140. Infarct sizes at 7 days were larger than at 3 days. The additive effect of multiple inhibitors was absent at 7 days. Ramiprilat and cFP-F-pAB significantly increased tissue BK levels. We conclude that inhibition of BK-inactivating enzymes protects endogenous BK from degradation and provides long-lasting protection from myocardial ischemia/reperfusion injury. A single treatment at the time of reperfusion does not prevent extension of the infarction between 3 and 7 days.     

Tumor necrosis factor type alpha stimulates human endothelial cells to produce granulocyte/macrophage colony-stimulating factor.

Tumor necrosis factor type alpha (TNF-alpha) is produced by monocytes and has been purified, sequenced, and cloned from the HL-60 cell line. Soluble products of monocytes stimulate endothelial cells to release multilineage hematopoietic colony-stimulating activity. To determine whether TNF-alpha could stimulate endothelial cells to produce these activities, we added recombinant human TNF-alpha to cultured human umbilical vein endothelial cells. Untreated endothelial cell conditioned medium and TNF-alpha-stimulated endothelial cell conditioned medium were tested for hematopoietic colony stimulating activity in colony-forming assays in methylcellulose. TNF-alpha stimulated growth factor production by endothelial cells. Fifth-passage human endothelial cells and multiply-passaged bovine aortic endothelial cells responded similarly to first-passage endothelial cells, indicating that the action of TNF-alpha on endothelial cells is direct and not due to contaminating lymphocytes or monocytes present in the first-passage cultures. To investigate the molecular basis for these findings, polyadenylylated RNA was prepared from the TNF-alpha-stimulated endothelial cells and probed for granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor mRNA. Granulocyte-macrophage colony-stimulating factor, but not granulocyte colony-stimulating factor, message was detected. This finding suggests that at least some of the hematopoietic colony-stimulating activity released by the TNF-alpha-stimulated endothelial cells is granulocyte-macrophage colony-stimulating factor. These results demonstrate that a purified monocyte product can stimulate endothelial cells to produce the multilineage growth factor granulocyte-macrophage colony-stimulating factor and extend the role of this immunoregulatory protein to the regulation of hematopoiesis in vitro.     

Absence of abnormalities of c-kit or its ligand in two patients with Diamond-Blackfan anemia.

As Diamond-Blackfan anemia shares clinical features with W and Steel defects in mice, we investigated the possibility that this human disorder might result from an abnormality of the c-kit receptor or its ligand, stem cell factor (SCF). For these studies, full nucleotide sequences for coding regions of c-kit and SCF were generated for two Diamond-Blackfan anemia patients and were normal. Similarly, the kds of SCF receptors on their marrow cells (31 pmol/L, 43 pmol/L) were comparable with those found in three normal controls (50 pmol/L, 55 pmol/L, 27 pmol/L). Serum SCF concentrations were 6.9 ng/mL in patient A, 14.6 ng/mL in patient B, who has been in hematologic remission since adolescence, and 2.7 ng/mL in the 3-year-old daughter of patient B, who also has Diamond-Blackfan anemia but is transfusion-dependent. It is possible that the SCF level in patient B increased with puberty, leading to her remission. These data provide evidence that Diamond-Blackfan anemia does not result from structural abnormalities of c-kit or SCF.     

Prognostic value of programmed electrical stimulation in patients with a recent episode of unstable angina

Patients with a recent episode of unstable angina have a 10% 1-year risk of sudden cardiac death. To determine prospectively whether electrophysiologic testing might be useful in predicting sudden death, 20 patients admitted to our hospital underwent programmed electrical stimulation as part of their evaluation. None had persistent angina, severe congestive heart failure, or sustained arrhythmias at the time of testing. Because of their long-term benefits, beta-blocking agents were continued whenever possible (18 of 20 patients). Ten of 20 patients (50%) had inducible ventricular tachycardia. In 19.5 months' mean follow-up, three patients (15%) either died suddenly or survived an episode of ventricular fibrillation. Programmed electrical stimulation was an insensitive (33%) and nonspecific (47%) predictor of sudden death in these patients. Programmed ventricular stimulation soon after admission for unstable angina is not a useful prognostic indicator for sudden death. Such patients do have a frequent induction of ventricular arrhythmias which appears to be a nonspecific marker of underlying coronary disease.