Synergistic effects of thrombopoietin and granulocyte colony-stimulating factor on neutrophil recovery in myelosuppressed mice.

Severe suppression of the hematopoietic system is a major factor in limiting chemotherapy dose escalation. To determine whether a combination of human recombinant granulocyte colony-stimulating factor (G-CSF) and thrombopoietin (TPO) would alter recovery of platelets, red blood cells (RBCs), or neutrophils after myeloablative therapy, myelosuppressed mice were treated with sc injections of TPO (90 micrograms/kg), G-CSF (250 micrograms/kg). TPO plus G-CSF or vehicle and complete blood counts were measured. Marrow and spleen cells were obtained at various times and assayed for erythroid, myeloid, and megakaryocytic progenitors. The prolonged neutropenia in vehicle controls (14 days) was significantly shortened in mice treated with G-CSF or TPO for 14 days. The combination of TPO plus G-CSF further reduced the duration of neutropenia. TPO and TPO plus G-CSF treatments also significantly shortened thrombocytopenia compared to vehicle. Recovery of RBCs was also enhanced in mice treated with either G-CSF or TPO, or the combination. Furthermore, treatment with G-CSF and/or TPO hastened myeloid, erythroid, and megakaryocyte progenitor recovery compared to vehicle controls. These results show that the combination of TPO plus G-CSF acts synergistically to accelerate neutrophil recovery in myelosuppressed mice and does not compromise the platelet or RBC response to TPO therapy.

Thrombopoietin accelerates platelet, red blood cell, and neutrophil recovery in myelosuppressed mice.

The recent cloning of thrombopoietin (TPO) has allowed us to study its in vivo effects in normal and myelosuppressed mice. Normal Balb/c mice were treated with recombinant human TPO (hTPO) at doses ranging from 1 to 20 kU for 7 days, and complete blood counts (CBCs) and the number of megakaryocytes in the bone marrow were determined. Platelet counts were increased starting on day 5 after mice were treated with hTPO. Platelet counts reached a peak between days 8 and 11 and returned to baseline between days 16 and 20. hTPO treatment increased the number of megakaryocytes in the bone marrow starting on day 3. In normal mice, hTPO treatment did not affect red or white blood cell (RBC or WBC) counts. To test the effects of hTPO in myelosuppressed mice, Balb/c mice were irradiated with 350 cGy total-body irradiation and dosed with 1.2 mg carboplatin, resulting in severe and prolonged thrombocytopenia, anemia, and neutropenia. Treatment with 5-20 kU hTPO for 7 days accelerated the recovery of platelet, RBC, and neutrophil counts in myelosuppressed mice and also significantly improved their nadirs. In addition, bone marrow megakaryocyte numbers recovered 11 days earlier and reticulocyte counts recovered 10 days earlier in hTPO-treated myelosuppressed mice than in controls. These results indicate that TPO can improve hematopoietic recovery in myelosuppressed mice, affecting multiple cell lineages.

Thrombopoietin expands erythroid, granulocyte-macrophage, and megakaryocytic progenitor cells in normal and myelosuppressed mice.

Thrombopoietin (Tpo), the ligand for the proto-oncogene receptor c-Mpl, increases megakaryocyte size, ploidy, and surface expression of platelet-specific glycoproteins, is inversely related to platelet mass, and is a potent in vivo stimulus of platelet production. However, several features of c-mpl biology, and that of its viral counterpart v-mpl, suggest that the action of Tpo may not be strictly limited to megakaryocytopoiesis. To investigate the possibility that Tpo might affect a multitude of cell lineages, we studied the effects of in vivo administration of the hormone on multiple types of marrow and splenic clonogenic hematopoietic progenitors. We report that Tpo acts to expand BFU-E, CFU-GM, and CFU-Mk and redistribute CFU-E in normal mice and to hasten the recovery of all of these progenitor cell types in myelosuppressed animals. These findings argue that the hematopoietic progenitor cell compartment responds to Tpo as a whole and that the in vivo effects of Tpo administration may be more wide-ranging than previously anticipated.

Platelet-derived growth factor and wound contraction in the rat.

This experiment was undertaken for three purposes: (1) to determine a dose-response curve of acute steroid inhibition of wound contraction in the rat; (2) to confirm the results of our preliminary study that platelet-derived growth factor (PDGF) enhanced wound contraction in acutely steroid impaired rats; and (3) to examine the histology of the PDGF-treated wounds. To determine the dose-response of acute steroid inhibition of wound contraction, the rats were suppressed with daily doses of methylprednisolone and wound contraction was measured. Results demonstrated that significant glucocorticoid-induced inhibition of wound contraction begins with daily methylprednisolone doses of 2.0 mg/wound/day or 6.7 mg/kg/day. In an effort to confirm the results of our previous study of the effect of PDGF on wound contraction in acutely steroid-impaired rats and to study the histology of the PDGF-treated wounds, rats were suppressed with methylprednisolone or hydrocortisone and administered daily topical doses of rPDGF-BB. Wound contraction measurements revealed no improvement in the amount or rate of wound contraction. Histologically, the wounds were all very similar in the patterns of cellularity, granulation tissue maturity, collagen content, and epithelial migration. We have clarified the dose response of acute steroid inhibition of wound contraction in rats, data previously unavailable, and have concluded that PDGF in reasonable doses does not improve wound contraction in steroid-impaired rats nor does it alter the histology of the wounds.

Murine thrombopoietin mRNA levels are modulated by platelet count.

The activity of the c-Mpl ligand hematopoietic progenitors meets criteria expected for thrombopoietin (TPO). Bio-assays have shown that blood TPO levels are inversely related to platelet mass. We sought to identify the molecular basis for this regulation. To determine if TPO mRNA levels respond to platelet demand, RNA from selected organs of mice with high, normal or low platelet counts was subjected to semiquantitative reverse transcriptase-polymerase chain reaction. Although no differences in TPO mRNA levels between control and treated mice could be detected in liver or kidney, TPO-specific bands were more intense after 25 to 30 polymerase chain reaction cycles in marrow-derived mRNA from thrombocytopenic mice. The TPO-specific bands were less intense in thrombocytotic mouse marrow and spleen than control mouse marrow and spleen after 30 cycles. These data support the hypothesis that TPO levels are regulated, at least in part, by modulating mRNA levels in response to platelet demand.

Thrombopoietin expands erythroid progenitors, increases red cell production, and enhances erythroid recovery after myelosuppressive therapy.

Thrombopoietin (TPO), the ligand for the receptor protooncogene c-mpl, has been cloned and shown to be the critical regulator of platelet production. Several features of c-Mpl expression, including its presence on erythroid cell lines, and the panmyeloid transformation characteristic of myeloproliferative leukemia (MPL) viral disease led us to investigate whether this receptor-ligand system may play a role in erythropoiesis. We report that although TPO alone did not support the growth of either early or late erythroid progenitors, it acted in synergy with erythropoietin to expand these populations. Moreover, while the effects on erythropoiesis in normal animals were modest, TPO greatly expanded the number of erythroid progenitors and blood reticulocytes and was associated with accelerated red cell recovery in myelosuppressed mice. Together, these data strongly suggest that erythroid progenitors respond to TOP and that this newly cloned cytokine, critical for platelet production, can augment erythropoiesis in states of marrow failure.

Inhibition of neointimal smooth muscle accumulation after angioplasty by an antibody to PDGF.

Approximately 30 to 40 percent of atherosclerotic coronary arteries treated by angioplasty or by bypass surgery occlude as a result of restenosis. This restenosis is due principally to the accumulation of neointimal smooth muscle cells, which is also a prominent feature of the advanced lesions of atherosclerosis. The factors responsible for the accumulation of intimal smooth muscle cells have not been identified. Platelet-derived growth factor (PDGF) is a potent smooth muscle chemoattractant and mitogen. It is present in platelets and can be formed by endothelium, smooth muscle, and monocyte-derived macrophages. The development of an intimal lesion in the carotid artery of athymic nude rats induced by intraarterial balloon catheter deendothelialization was inhibited by a polyclonal antibody to PDGF. These data demonstrate that endogenous PDGF is involved in the accumulation of neointimal smooth muscle cells associated with balloon injury and may be involved in restenosis after angioplasty, and perhaps in atherogenesis as well.

PDGF and FGF stimulate wound healing in the genetically diabetic mouse.

To examine the effects of recombinant growth factors in vivo, impaired wound healing was studied in genetically diabetic C57BL/KsJ-db/db mice. Large full-thickness skin wounds made on the backs of these mice exhibited significant delays in the entry of inflammatory cells into the wound, the formation of granulation tissue, and in wound closure when compared to their nondiabetic littermates. Recombinant human platelet-derived growth factor (rPDGF-BB, 1 or 10 micrograms), recombinant human basic fibroblast growth factor (rbFGF, 1 micrograms), or combinations of both were applied topically to the wounds for 5 to 14 days after wounding. Diabetic mouse wounds treated with rPDGF-BB or rbFGF had many more fibroblasts and capillaries in the wound bed at 10 and 21 days than did wounds treated with the vehicle alone. The animals treated with growth factors also had significantly greater wound closure at 21 days than those treated with the vehicle. Combinations of rPDGF-BB and rbFGF improved all parameters of healing but not to a greater extent than either growth factor alone. The effectiveness of rPDGF-BB and rbFGF suggest that recombinant growth factors may be useful in the treatment of patients with deficient wound repair.

Relative platelet-derived growth factor receptor subunit expression determines cell migration to different dimeric forms of PDGF.

Platelet-derived growth factor (PDGF) receptor transfectants of a fibroblastoid cell line (BHK) have been used to investigate the ability of the three dimeric forms of PDGF to elicit a chemotactic response. Cells transfected with the beta receptor subunit were only responsive to PDGF-BB, whereas cells expressing the alpha-receptor subunit were equally responsive to all three dimeric forms, PDGF-AA, PDGF-AB, and PDGF-BB. A positive chemotactic response correlated with rearrangement of actin organization. In a study of human arterial smooth muscle cells that express both PDGF receptor subunits endogenously, we again found that recombinant PDGF-AA could elicit a chemotactic response. However, the two smooth muscle cell isolates we examined differed in their chemotactic response to PDGF-AA. This difference correlated closely with their ability to respond mitogenically to this PDGF dimeric form, and the magnitude of both chemotactic and mitogenic responses was related to the proportion of the two receptor subunit species at the cell surface.

Pulmonary hypertension due to monocrotaline pyrrole is reduced by moderate thrombocytopenia.

To elucidate further the role of the platelet in the development of monocrotaline pyrrole (MCTP)-induced lung injury and pulmonary hypertension, MCTP-treated rats were made thrombocytopenic by cotreatment with an anti-rat platelet serum (PAS). Lung injury was assessed from increases in lung weight, lavage fluid protein concentration, and lactate dehydrogenase activity and from accumulation in lung tissue of 125I-labeled albumin. These indexes of injury were not different in MCTP-treated rats with normal or reduced platelet numbers at day 4,8, or 14. In MCTP-treated rats not receiving the PAS, pulmonary arterial pressure was elevated by day 8. However, pulmonary arterial pressure was the same as controls at both day 8 and day 14 in MCTP-treated rats made moderately thrombocytopenic by cotreatment with PAS. More marked reduction of platelet number abolished the protective effect of thrombocytopenia against pulmonary hypertension. In a separate series of experiments, treatment with antibodies to platelet-derived growth factor (PDGF), a potential mediator in the response to MCTP-induced injury, did not protect rats from the cardiopulmonary effects of MCTP. These data indicate that moderate reduction of the number of circulating platelets prevents MCTP-induced pulmonary hypertension but not MCTP-induced lung injury, suggesting that the platelet is involved in the pulmonary hypertensive response to MCTP-induced lung injury by unknown mechanisms.

Effects of growth factors in vivo. I. Cell ingrowth into porous subcutaneous chambers.

Growth factors secreted by platelets and macrophages may play roles in atherogenesis and in wound repair. The multiple biologic effects of these factors are being studied extensively in vitro, but their roles in vivo are relatively unexplored. The cellular responses to platelet-derived growth factor (PDGF), transforming growth factor beta (TGF beta), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF) were examined in a wound chamber model in rats. Growth factors were emulsified in bovine dermal collagen suspensions, placed in 1 X 30-mm porous polytetrafluoroethylene tubes, inserted subcutaneously, and removed after 10 days. The presence of PDGF (400 ng), TGF beta (200 ng), or bFGF (100 ng) increased the DNA content of the chambers two- to sixfold, compared with controls. Regardless of dose, EGF (100-800 ng) did not affect the DNA content. The increases in DNA observed for PDGF, TGF beta, or bFGF resulted from accumulations of varying numbers of fibroblasts, capillaries, macrophages, and leukocytes in 10-day chambers. The addition of 250 micrograms/ml heparin to the collagen suspension potentiated the response to PDGF and bFGF, but not to TGF beta or EGF. The clearance of 125I-labeled growth factors from the chambers was biphasic. After an initial rapid phase, the remaining growth factor was slowly cleared. The half-life of the initial phase was rapid for PDGF (12 hours) and bFGF (9 hours) and somewhat slower for TGF beta (22 hours). There was no difference in the rate of clearance between collagen and collagen/heparin matrices for any of the growth factors examined. These studies demonstrate that PDGF, bFGF, and TGF beta can induce granulation tissue development in normal animals. The similarity in cellular responses to three peptides with differing in vitro actions suggests that the responses observed at 10 days reflect a secondary process, possibly mediated by effector cells such as macrophages, lymphocytes, or granulocytes that are attracted into the chamber by each growth factor, rather than a direct effect of the factors themselves.

Monocrotaline pyrrole-induced cardiopulmonary toxicity is not altered by metergoline or ketanserin.

Monocrotaline pyrrole (MCTP) causes endothelial cell damage, pulmonary hypertension and right ventricular hypertrophy in rats by an undetermined mechanism. A role for 5-hydroxytryptamine (5-HT) in the cardiopulmonary response to MCTP has been suggested. To investigate the role of 5-HT, the effects of two 5-HT receptor antagonists were examined in MCTP-treated rats. Cotreatment with metergoline, an antagonist which binds to both 5-HT1 and 5-HT2 receptors, did not alter MCTP-induced elevation of lung weight or right ventricular hypertrophy. 5-HT-induced vascular smooth muscle contractions are mediated by 5-HT2 receptors; therefore, MCTP-treated rats were cotreated with ketanserin (KET), a specific 5-HT2 receptor antagonist. At a dosing regimen of KET that inhibited the 5-HT-induced platelet shape change in platelet-rich plasma and the 5-HT-induced increase in perfusion pressure in isolated lungs, KET did not affect the elevation in lung weight or the increased accumulation of 125I-albumin in the lung tissue of MCTP-treated rats. Moreover, MCTP-induced right ventricular hypertrophy was not attenuated by KET. These results indicate that cotreatment with either of these two 5-HT receptor antagonists does not alter the lung injury or right ventricular hypertrophic response to MCTP and suggest that 5-HT is not necessary for MCTP-induced toxicity.