The role of transforming growth factor‐β in PEG‐rHuMGDF‐induced reversible myelofibrosis in rats

Abstract

Summary. Pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) injected at a suprapharmacologic dose (100 mg/kg) daily for 5 d in normal rats caused marked increases in marrow megakaryocytes and platelet counts at 6-8 d followed by gradual decreases to control levels at 10-20 d. Interestingly, in addition to the expected thrombopoiesis, PEG-rHuMGDF was associated with myelofibrosis with a predominance of reticulin fibres at day 10 followed by complete normalization by day 20. At 6-8 d, the levels of transforming growth factor-b1 (TGF-b1) in the extracellular fluid of the marrow, the platelet poor plasma, and the platelet extract were increased 23-, 7-and 2-fold, respectively. The elevated levels of TGF-b1 were gradually reduced to baseline levels at 13-20 d in accordance with the normalization of myelofibrosis and thrombopoiesis. An ultrastructural analysis showed that large fragments of megakaryocytes were deposited in the marrow parenchyma of PEG-rHuMGDF-treated rats at day 6. PEG-rHuMGDF administration at pharmacologic doses (1 and 10 mg/kg) did not induce the deposition of reticulin fibres in the marrow. These findings suggest that TGF-b1 leaked from megakaryocytes is involved in the development of the PEG-rHuMGDF-induced myelofibrosis and that this is a reversible process related to the regulation of the excess production of platelets.

Myelofibrosis with Myeloid Metaplasia: New Developments in Pathogenesis and Treatment

“…While megakaryocytes from patients with MMM can grow in the absence of TPO, they are still responsive to the effect of the cytokine (20). These observations prompted the development of models of the disease whereby laboratory animals are exposed to chronically elevated levels of TPO either administered exogenously or continuously by gene transfer using vectors (21)(22)(23)(24). These mice develop a bone marrow failure syndrome similar to human MMM and die within a few months due to progressive disease.…”

“…TGF-1 release is temporally related to TPO stimulation and marrow fibrosis does not occur in TPO stimulated mice if they do not express TGF-1 (22,27). Osteoprotegerin is an important inhibitor of oseoclastogenesis and animals engineered to overexpress OPG develop osteopetrosis (28).…”

Prominent role of TGF-β1 in thrombopoietin-induced myelofibrosis in mice

“…[21][22][23] In these experimental models, it has been hypothesized that TGF-␤1 had an important role in the development of myelofibrosis because elevated levels were detected in plasma, platelet extracts, and marrow fluids at the time of myelofibrosis development, which gradually decreased with fibrosis normalization. 20 However, increased levels of PDGF were also demonstrated in plasma from TPO-overexpressing mice, 22 suggesting that both TGF-␤1 and PDGF, or possibly other cytokines, could influence the fibrogenic and osteogenic responses.…”

“…Samples were prepared with a slight modification of the reported procedure. 20 Briefly, 500 L whole blood was collected on 500 L citrated Hanks buffered saline solution (HBSS, Sigma Aldrich) and centrifuged at 200g to prepared platelet-rich plasma. Platelets were pelleted (2000g for 10 minutes), suspended in 200 L HBSS, and counted.…”

“…In vivo administration of suprapharmacologic doses of thrombopoietin (TPO), the physiological regulator of platelet production, resulted in megakaryocyte hyperplasia associated with a densification of the reticulin network. 19,20 Mice permanently exposed to high doses of TPO delivered through retroviral infection of hematopoietic stem cells developed a myeloproliferative syndrome with a prominent proliferation of megakaryocytes and leukocytes, extramedullary hematopoiesis and, invariably, splenic and medullary fibrosis and osteosclerosis. [21][22][23] In these experimental models, it has been hypothesized that TGF-␤1 had an important role in the development of myelofibrosis because elevated levels were detected in plasma, platelet extracts, and marrow fluids at the time of myelofibrosis development, which gradually decreased with fibrosis normalization.…”