The Ultrastructure Features of the Advanced Stages of Fibrosis in GATA-1low Mice Are Similar to Those Found in Wound Healing.
Authors: Zingariello Maria , Ghinassi Barbara , Rana Rosa Alba , Lorenzini Lorenzo R.N. , Vannucchi Alessandro M. , Paoletti Francesco , Migliaccio Anna Rita F. 
Published: October 2019

Abstract
The fibrosis associated with idiopatic myelofibrosis (IM) is thought to be a reactive process mediated by cytokines released by abnormal megakaryocytes (MKs) in the microenviroment. The biochemical details of this process are, however, still not known. GATA-1low mice develop with age myelofibrosis, a syndrome very similar to IM, that manifests itself with thrombocytopenia, anemia and teardrop poikilocytes in the blood and marrow and spleen fibrosis at 12-month of age. From 15-month-on, extramedullary hematopoiesis is observed in the liver. Electron microscopy studies have shown that MKs from GATA-1low mutants are blocked between stage I and II of maturation. The block includes failure to organize the α-granules, abnormal P-selectin localization on the Demarcation Membrane System (DMS), and pathological neutrophil emperipolesis. The neutrophils embedded in the MKs release proteases that are thought to mediate release of TGF-β in the microenviroment through the canaliculi of the DMS. TGF- β is supposed, then, to activate the fibroblasts to produce fibers (Centurione et al, Blood104:3573, 2004). To clarify the relationship between MKs, neutrophil emperipolesis and fibroblast activation, we present here the ultrastructural analysis of the spleen during the disease progression of GATA-1low mice. The animals were divided into three age groups: 5–8-month (IM-free), 10–12-month (pre-myelofibrotic) and 15-month-on (myelofibrotic). As disease progressed, the number of TGF-β-related immunogold-particles increased from ~30 (IM-free) to 80 (pre-myelofibrotic) and 230 (fibrotic) per field in all of the cells anlyzed (MKs, neutrophils and fibroblasts). TGF-β was found equally localized in the cytoplasm and in nucleus of these cells. In the fibroblasts, the rise of TGF-β content was associated with a dramatic change in morphology. In fact, the fibroblasts from old animals, envolved into larger cells with long protrutions that surrounded the MKs. The link between these protrusions and the MKs were so tight that at times the all structure appeared as a thickening of the cytoplasmic membrane of the MK. Numerous TGF-β particles we found located along these protrutions. Furthermore, some of the fibroblasts from these old animals acquired the morphology of myofibroblasts, with several myosin-like structures in the cytoplasm. These myofibroblasts were localized around the MK nests and isolated these nests from the surrounding fibrotic areas. On the other hand the fibrotic areas were characterized by the presence, in addition to the expected collagen fibers, of muscle fibers intercrossing each other. The presence of high TGF-β content within the cells, the differentiation of fibroblasts into myofibroblasts, the close contact of these cells with MK, as well as the presence of myosin fibers in the connective areas, are all features found also in the process of would healing and in the fibrosis associated with fibrocontractive disease. In summary, these results indicate that abnormal MKs, in addition to pathological interactions with neutrophils, establish pathological interactions with other cell types, including the fibroblasts. We suggest that these interactions may contribute to alter the microenviroment milleu of old GATA-1low mice.