The mechanisms by which megakaryocytes (MKs) differentiate and release platelets into the circulation are not well understood. However, growing evidence indicates that a complex regulatory mechanism involving MK-matrix interactions may contribute to the quiescent or permissive microenvironment related to platelet release within bone marrow. To address this hypothesis, in this study we demonstrate that human MKs express and synthesize cellular fibronectin (cFN) and transglutaminase factor XIII-A (FXIII-A). We proposed that these 2 molecules are involved in a new regulatory mechanism of MK-type I collagen interaction in the osteoblastic niche. In particular, we demonstrate that MK adhesion to type I collagen promotes MK spreading and inhibits pro-platelet formation through the release and relocation to the plasma membrane of cFN. This regulatory mechanism is dependent on the engagement of FN receptors at the MK plasma membrane and on transglutaminase FXIII-A activity. Consistently, the same mechanism regulated the assembly of plasma FN (pFN) by adherent MKs to type I collagen. In conclusion, our data extend the knowledge of the mechanisms that regulate MK-matrix interactions within the bone marrow environment and could serve as an important step for inquiring into the origins of diseases such as myelofibrosis and congenital thrombocytopenias that are still poorly understood. (Blood. 2011;117(8):2476-2483) IntroductionHemopoietic stem cells reside in bone marrow-specialized niches that dictate how they differentiate, proliferate, mature, and enter the peripheral circulation. [1][2][3][4] Megakaryocyte (MK) maturation and platelet generation are consequent to MK migration from the osteoblastic to the vascular niche, where MKs extend pro-platelets and newly generated platelets are released into the bloodstream. 5,6 The characteristics of the microenvironment surrounding MKs play an important role in the regulation of platelet production within the bone marrow. 3,7 In particular, the interaction of MKs with different extracellular matrices (ECMs) that fill the bone marrow spaces seems to orchestrate their maturation in specific sites. 8 It has been demonstrated that interactions of primary human MKs with matrices thought to fill the vascular niche, such as fibrinogen or von Willebrand factor, are able to sustain MK maturation and pro-platelets, whereas type I collagen totally suppresses these events and prevents premature platelet release in the osteoblastic niche. 7,9 The negative regulation of pro-platelets by type I collagen is mediated by the interaction with the integrin ␣2␤1 and involves the Rho/ROCK pathway. 10,11 However, the exact sequence of events that determines the interaction of MKs with the ECM, and therefore their regulation, is not completely understood. 12 Recent studies 13 have demonstrated that the encounter between a cell and an adhesive substrate involves an initial passive interaction characterized by cell adhesion and spreading, followed by an active stage that involves actin polymerization and...

Collagen fibrils were obtained in vitro by aggregation from acid-soluble type I collagen at different initial concentrations and with the addition of decorin core or intact decorin. All specimens were observed by scanning electron microscopy and atomic force microscopy. In line with the findings of other authors, lacking decorin, collagen fibrils undergo an extensive lateral association leading to the formation of a continuous three-dimensional network. The addition of intact decorin or decorin core was equally effective in preventing lateral fusion and restoring the normal fibril appearance. In addition, the fibril diameter was clearly dependent on the initial collagen concentration but not on the presence/absence of proteoglycans. An unusual fibril structure was observed as a result of a very low initial collagen concentration, leading to the formation of huge, irregular superfibrils apparently formed by the lateral coalescence of lesser fibrils, and with a distinctive coil-structured surface. Spots of incomplete fibrillogenesis were occasionally found, where all fibrils appeared made of individual, interwined subfibrils, confirming the presence of a hierarchical association mechanism.

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Cell interactions with matrices via specific receptors control many functions, with chemistry, physics, and membrane elasticity as fundamental elements of the processes involved. Little is known about how biochemical and biophysical processes integrate to generate force and, ultimately, to regulate hemopoiesis into the bone marrow-matrix environment. To address this hypothesis, in this work we focus on the regulation of MK development by type I collagen. By atomic force microscopy analysis, we demonstrate that the tensile strength of fibrils in type I collagen structure is a fundamental requirement to regulate cytoskeleton contractility of human MKs through the activation of integrin-␣2␤1-dependent Rho-ROCK pathway and MLC-2 phosphorylation. Most importantly, this mechanism seemed to mediate MK migration, fibronectin assembly, and platelet formation. On the contrary, a decrease in mechanical tension caused by N-acetylation of lysine side chains in type I collagen completely reverted these processes by preventing fibrillogenesis. (Blood. 2011;118(16):4449-4453) IntroductionCollagen fibrils are the main architectural element in several tissues and bones, and the self-assembly processes involved in collagen fibrillogenesis are of enormous importance to matrix pathology and proper cell development. 1 Type I collagen plays a critical role in the bone marrow niche environment, preventing platelet formation by MKs through the activation of integrin-␣2␤1 and the Rho-ROCK pathway. 2,3,4 Changes in mechanical properties are tightly coupled in vivo to changes in matrix composition, crosslinking, and density. 5 Moreover, different signaling pathways, related to the transduction of mechanical signals into biochemical responses, regulate cell behavior. 6 However, the question on how biochemical and biophysical processes integrate to generate force and, ultimately, to regulate MK fate, is still open. Therefore, our purpose for the present work was to focus on MK function related to type I collagen organization and biomechanics. MethodsType I collagen from bovine tendon was modified as previously described 9 and as reported in supplemental Methods (available on the Blood Web site; see the Supplemental Materials link at the top of the online article). Antibodies and solid-phase binding assay are described in supplemental Methods. MKs were differentiated from human cord blood progenitors as previously described as well as proplatelet and spreading assay, electron and immunofluorescence microscopy, second harmonic generation, and Western blotting analysis. 4,7,8 Human cord blood was collected on informed consent of the parents, with approval from the ethical committee of the Istituto di Ricovero e Cura a Carattere Scientifico Policlinico San Matteo Foundation in accordance with the principles of the Declaration of Helsinki. Binding of cells to different collagen preparations was evaluated with 25 ϫ 10 3 cells seeded for 1 hour in Stem Span medium (Stem Cell Technologies) in a 96-well plate precoated with 5 g of each collagen prepa...

Nonalcoholic fatty liver disease (NAFLD) is a serious health problem in developed countries. We documented the effects of feeding with a NAFLD-inducing, methionine- and choline-deficient (MCD) diet, for 1–4 weeks on rat liver oxidative stress, with respect to a control diet. Glycogen, neutral lipids, ROS, peroxidated proteins, and SOD2 were investigated using histochemical procedures; ATP, GSH, and TBARS concentrations were investigated by biochemical dosages, and SOD2 expression was investigated by Western Blotting. In the 4-week-diet period, glycogen stores decreased whereas lipid droplets, ROS, and peroxidated proteins expression (especially around lipid droplets of hepatocytes) increased. SOD2 immunostaining decreased in poorly steatotic hepatocytes but increased in the thin cytoplasm of macrosteatotic cells; a trend towards a quantitative decrease of SOD expression in homogenates occurred after 3 weeks. ATP and GSH values were significantly lower for rats fed with the MCD diet with respect to the controls. An increase of TBARS in the last period of the diet is in keeping with the high ROS production and low antioxidant defense; these TBARS may promote protein peroxidation around lipid droplets. Since these proteins play key roles in lipid mobilization, storage, and metabolism, this last information appears significant, as it points towards a previously misconsidered target of NAFLD-associated oxidative stress that might be responsible for lipid dysfunction.