Cigarette smoke exposure up-regulates endothelin receptor B in human pulmonary artery endothelial cells: molecular and functional consequences

BACKGROUND AND PURPOSE

Pulmonary arteries from smokers and chronic obstructive pulmonary disease patients show abnormal endothelium-dependent vascular reactivity. We studied the effect of cigarette smoke extract (CSE) on endothelin receptor B (ET_B) expression in human pulmonary artery endothelial cells (HPAECs) and its role in endothelial dysfunction.

EXPERIMENTAL APPROACH

ET_B receptor expression was measured by real time RT-PCR, Western blot and immunofluorescence. Cell contraction, intracellular Ca²⁺, F/G-actin, RhoA activity, myosin light chain phosphorylation, ET, NO, thromboxane (Tx)A₂ and reactive oxygen species (ROS) were measured by traction microscopy, fluorescence microscopy, phalloidin fluorescence, colorimetric assay, Western blot, elisa and DCFDA fluorescence respectively.

KEY RESULTS

Cigarette smoke extract dose-dependently increased ET_B receptor expression in HPAECs after 24 h incubation. CSE-induced ET_B expression was attenuated by bosentan, the ET_B receptor antagonist BQ788, the Rho kinase antagonist Y27632 and the antioxidant N-acetylcysteine. A monoclonal antibody to ET-1 prevented CSE-induced ET_B receptor overexpression. Twenty-four hour exposure to ET-1 dose-dependently increased ET_B receptor expression, mimicking the effect of CSE. CSE-induced ET_B receptor overexpression caused greater cell contraction; increased intracellular Ca²⁺; increased F/G-actin and RhoA activity; increased myosin light chain phosphorylation; augmented TxA₂ and ROS production; and decreased NO after acute ET-1 (10 nM). These effects were attenuated by bosentan, BQ788, Y27632 and N-acetylcysteine.

CONCLUSIONS AND IMPLICATION

Cigarette smoke extract induced ET_B receptor overexpression by a feed forward mechanism mediated partly by ET release, promoting HPAEC dysfunction and attenuated by ET_B receptor blockade, Rho kinase and ROS inhibition. These results provide support for the use of bosentan in CS-related endothelial dysfunction.     

Clonal dysregulation of the antibody response to tetanus-toxoid after bone marrow transplantation

After bone marrow transplantation (BMT), a prolonged dysregulation of humoral immunity can be observed. In the present study, we investigated whether this is reflected in an abnormal production of specific antibodies (Ab) to the T-cell-dependent recall antigen tetanus-toxoid (TT). The study group consisted of children receiving transplants of an unmodified allogeneic graft and of adults receiving either a T-cell- depleted allogeneic or an unmodified autologous BM graft. Findings were compared with those in healthy controls. In pediatric graft recipients, who were routinely revaccinated early after BMT, the Ab response was quantitatively superior to that in adult graft recipients who did not receive early revaccination. In the majority of graft recipients, the time period after vaccination required to reach the peak level of antibodies was prolonged and the number of responding TT-specific B- cell clones was markedly decreased in comparison with controls. In controls, a low frequency of dominant B-cell clones may produce low quantities of homogeneous Ab components (H-Ab) against a heterogeneous background. However, in BM graft recipients, "overshooting" of Ab production by separate B-cell clones was observed, resulting in the development of H-Ab at a relatively high concentration. These abnormalities were present up to 10 years after BMT, irrespective of either the age of the recipient, the modulation of the graft, or the vaccination schedule used. It is hypothesized that the dysregulated Ab production is the consequence of activation of a restricted number of resting memory B cells, present in germinal centers, repopulating gradually after BMT. Our data show that routine revaccination early after BMT improves the humoral immune response. However, because of a clonally dysregulated Ab production, long-lasting qualitative defects may be present even after normalization of Ab titers.     

Demonstration of a blood-bone marrow barrier to macrophage colony- stimulating factor

Several previous studies suggested that murine macrophage colony- stimulating factor (CSF-1) might have impaired access to hematopoietic cells in the marrow. The apparent lack of hematopoietic responses to exogenous CSF and the finding of available or unoccupied CSF receptors despite saturating CSF levels in the serum led to studies of a potential blood-bone marrow barrier for this factor. Groups of mice were injected with pure unlabeled CSF-1 by either intravenous (IV) or intraperitoneal (IP) routes. Marrow and spleen cells were obtained at intervals after injection, held at 0 degree C, and assessed for changes in binding of 125I-CSF. Saturation of all available CSF receptors is achieved in vitro with 100 to 150 U CSF/mL. Despite achieving serum levels of 5,000 to 7,000 U/mL after IV injection of 25,000 units of CSF, less than 50% of the marrow receptors and less than 85% of the splenic receptors were saturated or downregulated. The decline in receptor availability was transient, with return of receptor sites in two to four hours. Increasing the IV dose to 125,000 units increased serum CSF values to approximately 20,000 U/mL and led to a virtual disappearance of available receptors for two to three hours. When administered IP, only approximately 40% of marrow and 80% of splenic receptors were affected for two hours. It was necessary to increase the dose of CSF to 250,000 units IP to saturate or downregulate receptors for three to four hours after injection. These observations indicate a marked blood-bone marrow barrier and lesser blood-spleen barrier for the transfer of serum CSF to responsive hematopoietic cells in vivo.     

Human interleukin-5 (IL-5) regulates the production of eosinophils in human bone marrow cultures: comparison and interaction with IL-1, IL-3, IL-6, and GMCSF

Recombinant human interleukin-5 (rhIL-5), in either liquid or semi- solid cultures, selectively induced eosinophil production from normal human bone marrow, with no activity on other cell lineages. The time course of eosinophil production induced by murine IL-5, rhIL-3, and rh granulocyte-macrophage colony stimulating factor (GMCSF) was similar to rhIL-5. The rate of eosinophil maturation in vitro was independent of the stimulating cytokine, mature eosinophils being produced after 4 to 5 weeks in liquid culture with each of these cytokines. The eosinophils produced in response to each cytokine were morphologically indistinguishable, and had the ultrastructural features of maturity except that the electron-dense material in the granules had not formed into crystalline cores. Neither rhIL-1 nor rhIL-6 alone, or in combination with rhIL-5 or rhIL-3, induced eosinophil differentiation or proliferation under the conditions used. rhIL-3 and rhGMCSF induced more eosinophil colonies than rhIL-5, rhIL-5 had an additive, not synergistic, effect on eosinophil colony production when combined with either rhIL-3 or rhGMCSF, suggesting that rhIL-5 stimulates a smaller and possibly different population of eosinophil progenitors. However, rhIL-5 induced the greatest eosinophil production in liquid cultures, suggesting that although it may act on a smaller population of precursors, it is able to stimulate more proliferative steps than either rhIL-3 or rhGMCSF.     

Constitutive expression of platelet glycoproteins by the human leukemia cell line K562

The human leukemia cell line K562 was derived from a patient with chronic granulocytic leukemia. This cell line has subsequently been shown to possess phenotypic markers typical of erythroid and myeloid cells. Using a rabbit antiserum directed against purified platelet glycoproteins (PGPs), we have obtained evidence for the constitutive expression of PGPs on the surface of K562 cells. PGPs expressed have been tentatively identified as IIa and III based on their apparent migration in a 7% sodium dodecylsulfate polyacrylamide gel. K562 may become an important tool for the study of early events involved in megakaryocytic differentiation.     

An investigation into the role of coagulation factor XIII in ADP- induced aggregation and fibrinogen binding with rabbit platelets

Because there was a possibility that activated factor XIII (factor XIIIa) might stabilize a platelet-fibrinogen aggregate through its crosslinking action, we have isolated plasma factor XIII, activated it, and studied the effect of factor XIIIa at a concentration of 3.3 micrograms/ml on aggregation and 125I-fibrinogen binding of rabbit platelets stimulated with 9 microM ADP. Factor XIIIa did not cause aggregation in the absence of ADP, nor did it enhance ADP-induced aggregation or substantially stabilize the platelet aggregate. The presence of factor XIIIa did not affect the amount of fibrinogen bound to platelets immediately after stimulation with ADP, but it appeared to cause a slow specific binding of 125I-fibrinogen to platelets whether or not they were stimulated with ADP. This binding, which was not inhibited by prostaglandin E1, did not lead to aggregation and was accompanied by crosslinking of fibrinogen through its A alpha and gamma chains, either to other fibrinogen molecules or to a platelet protein or proteins.     

Diaziquone given as a continuous infusion is an active agent for relapsed adult acute nonlymphocytic leukemia

Diaziquone given as a bolus has not been effective in patients with relapsed or refractory leukemia. Because of in vitro data suggesting enhancement of diaziquone-induced cytotoxicity for human and murine leukemia cells with increased duration of drug exposure and the relatively short terminal plasma half-life of diaziquone, 49 patients (34 acute nonlymphocytic leukemia [ANLL], six chronic myelogenous leukemia in blast crisis [CML-B], five acute lymphocytic leukemia [ALL], four 2 degrees ANLL) with leukemia were given diaziquone as a continuous infusion for seven days. The maximum tolerated dose was 28 mg/m2/d for seven days. The dose-limiting toxicity was the duration of bone marrow aplasia (median, 49 days to greater than 500 PMNs in responders; range, 28 to 101 days). Nonhematologic toxicity was minimal. Responses occurred only in patients with relapsed ANLL, of whom 26 were treated at effective doses. There were six complete responses (CR) (23%) and two partial responses (PR) (8%), although five of eight responders never achieved platelet counts greater than 100,000/microL. Thrombocytopenia in these patients was felt to be a manifestation of diaziquone effect, not persistence of leukemia. The median duration of CR was 195 days (range, 88 to 860+). One patient had active CNS leukemia at the start of treatment and has had a durable (28+ month) CR in both sites of disease. Diaziquone produced prolonged aplasia in patients with secondary ANLL and CML-B (five of ten patients died aplastic), whereas patients with ALL all had regrowth of leukemia and two failed to become aplastic. The lack of significant nonhematologic toxicity and the activity in patients with relapsed ANLL render diaziquone of interest as second-line therapy or consolidation therapy in first remission for patients with ANLL.     

Effects of the cell adhesion peptide, Arg-Gly-Asp-Ser, on responses of washed platelets from humans, rabbits, and rats

Fibrinogen is a cofactor in the aggregation of human platelets, and is required for ADP-induced aggregation of washed platelets; however, exogenous fibrinogen is not required for ADP-induced aggregation of washed platelets from rabbits or rats. Because with human platelets the cell adhesion peptide, Arg-Gly-Asp-Ser (RGDS), inhibits aggregation and the binding of 125I-fibrinogen to ADP-stimulated platelets, its effects on rabbit and rat platelets were studied to investigate the differences in the fibrinogen requirements of platelets from the three species. RGDS (50 mumol/L) caused greater than 80% inhibition of thrombin- induced or (ADP + fibrinogen)-induced aggregation of human platelets, but only 3% to 9% inhibition of the aggregation of rabbit or rat platelets, regardless of whether fibrinogen was added. RGDS inhibited the binding of 125I-fibrinogen to ADP-stimulated human platelets by 80% to 90%, but by only 15% to 27% in the case of rabbit or rat platelets. The differences were due to the species of platelets, since human and rabbit fibrinogens gave similar results. In addition, RGDS failed to displace fibrinogen from the surface of rabbit platelets that had been stimulated with ADP. Thus, there are species differences in the ability of the cell adhesion peptide, RGDS, to block the platelet fibrinogen receptor, even within the mammalian species.     

Amifostine (WR-2721) shortens the engraftment period of 4- hydroperoxycyclophosphamide-purged bone marrow in breast cancer patients receiving high-dose chemotherapy with autologous bone marrow support

4-Hydroperoxycyclophosphamide (4-HC), a commonly used marrow-purging agent, is active against many tumors, but is also toxic to normal marrow progenitors. Amifostine (WR-2721) is a sulfhydryl compound with chemoprotectant activity. Preclinical studies using suspensions of bone marrow and breast cancer cells demonstrated that ex vivo treatment with amifostine followed by 4-HC resulted in protection of marrow progenitors, with no compromise in the antitumor effect of 4-HC. This fact stimulated the development of a clinical trial. Bone marrow was harvested from 15 poor-prognosis breast cancer patients and randomly assigned to ex vivo treatment with amifostine followed by 4-HC (amifostine + 4-HC), or treatment with 4-HC alone. High-dose chemotherapy was then administered followed by infusion of the purged autologous bone marrow support (ABMS). Leukocyte engraftment, defined as a white blood cell count > or = 1 x 10(9)/L, was achieved in an average of 26 days for patients whose marrow was purged with amifostine + 4-HC versus 36 days for patients whose marrow was purged with 4-HC alone (P = .032). The average number of platelet transfusions (12 v 29; P = .017) and days of antibiotic therapy (28 v 40; P = .012) were significantly less for patients whose marrow was exposed to amifostine + 4-HC, compared with 4-HC alone. Unpurged backup marrow fractions were infused into three patients whose marrow was purged with 4-HC alone, because of inadequate marrow recovery. None of the patients who received amifostine + 4-HC-purged marrow required a backup marrow fraction. Complete remissions were achieved in 83% of patients with measurable disease, with no difference between the two cohorts. Forty- three percent of patients remained alive and progression-free at a mean of 13 months posttransplant. There was no significant difference in the rate or pattern of relapse for patients whose marrow was purged with amifostine + 4-HC compared with those whose marrow was purged with 4-HC alone. Ex vivo treatment of marrow with amifostine significantly shortens the time to marrow recovery, thereby reducing the risk of myelosuppressive complications in breast cancer patients receiving high- dose chemotherapy and 4-HC-purged ABMS. Since supportive care requirements are also significantly decreased, amifostine may reduce the cost of such therapy.