Megakaryocytes Contribute to the Bone Marrow-Matrix Environment by Expressing Fibronectin, Type IV Collagen, and Laminin

Abstract

Megakaryocytes associate with the bone marrow vasculature where they convert their cytoplasm into proplatelets that protrude through the vascular endothelium into the lumen and release platelets. The extracellular matrix (ECM) microenvironment plays a critical role in regulating these processes. In this work we demonstrate that, among bone marrow ECM components, fibronectin, type IV collagen and laminin are the most abundant around bone marrow sinusoids and constitute a peri-cellular matrix surrounding megakaryocytes. Most importantly, we report, for the first time, that megakaryocytes express components of the basement membrane and that these molecules contribute to the regulation of megakaryocyte development and bone marrow ECM homeostasis both in vitro and in vivo. In vitro, fibronectin induced a three-fold increase in the proliferation rate of mouse hematopoietic stem cells leading to higher megakaryocyte output with respect to cells treated only with thrombopoietin or other matrices. However, megakaryocyte ploidy level in fibronectin-treated cultures was significantly reduced. Stimulation with type IV collagen resulted in a 1.4-fold increase in megakaryocyte output, while all tested matrices supported proplatelet formation to a similar extent in megakaryocytes derived from fetal liver progenitor cells. In vivo, megakaryocyte expression of fibronectin and basement membrane components was up-regulated during bone marrow reconstitution upon 5-fluorouracil induced myelosuppression, while only type IV collagen resulted up-regulated upon induced thrombocytopenia. In conclusion, this work demonstrates that ECM components impact megakaryocyte behavior differently during their differentiation and highlights a new role for megakaryocyte as ECM-producing cells for the establishment of cell niches during bone marrow regeneration.

Proplatelet formation is selectively inhibited by collagen type I through Syk-independent GPVI signaling

“…In contrast to a previous study where collagen I was exclusively found in cortical bone and collagen IV was only associated with vessels (Nilsson et al, 1998), we readily also detected both types in the bone marrow cavity (Fig. 2) in line with a recent study (Malara et al, 2014). The discrepancy between these two recent studies and the study of Nilsson might be attributed to different decalcification methods, as harsh acid treatment can destroy epitopes (Athanasou et al, 1987).…”

“…It is well known that structural proteins present within the bone marrow cavity and at the vascular niche have an impact on thrombopoiesis (Larson and Watson, 2006). Several in vitro systems have been established to study PPF on plated extracellular matrix (ECM) substrates that include the use of cord-blood-derived CD34+ cells (Balduini et al, 2008;Malara et al, 2011;Sabri et al, 2004) and murine megakaryocytes from bone marrow or fetal liver (Malara et al, 2014). These studies suggest that, although platelet biogenesis is a cell-autonomous process, collagen type I inhibits PPF by preventing premature platelet release in the bone marrow cavity, whereas collagen type IV and laminin support PPF at the sinusoids (Balduini et al, 2008).…”

Quantification and three-dimensional microanatomical organization of the bone marrow

“…Studies in BM and other lymphoid organs indicate that highly plastic ECM networks could play vital roles in steady state as well as pathological hematopoiesis. 61,62 However, not much is known to date on the heterogeneity, biochemical composition, and topology of BM ECM.…”

“…Studies in BM and other lymphoid organs indicate that highly plastic ECM networks could play vital roles in steady state as well as pathological hematopoiesis. 61,62 However, not much is known to date on the heterogeneity, biochemical composition, and topology of BM ECM.Spatial organization of hematopoiesis. How the succession of orderly events that leads to continuous hematopoiesis develops within the described tissue microfabric remains poorly understood.…”

Extracellular molecules in hematopoietic stem cell mobilisation

“…CXCL12 is produced by a number of micro-environmental cells, including CXCL12-abundant reticular (CAR) cells [22], nestin+ cells [23], cells of the osteoid lineage [24], arteriolar pericytes [25], and endothelial cells [26]. Once produced, CXCL12 may be bound to matrix components, including fibronectin [27], collagen IV [28], and heparan sulfate [29] expressed within the niche [30,31] potentially refining the activity of this chemokine. Therefore, CXCL12 has the potential to facilitate the retention of HSC in multiple niches within the bone marrow.…”