Effects of protein malnutrition on hematopoietic regulatory activity of bone marrow mesenchymal stem cells.

Protein malnutrition causes anemia and leukopenia as it reduces hematopoietic precursors and impairs the production of mediators that regulate hematopoiesis. Hematopoiesis occurs in distinct bone marrow niches that modulate the processes of differentiation, proliferation and self-renewal of hematopoietic stem cells (HSCs). Mesenchymal stem cells (MSCs) contribute to the biochemical composition of bone marrow niches by the secretion of several growth factors and cytokines, and they play an important role in the regulation of HSCs and hematopoietic progenitors. In this study, we investigated the effect of protein malnutrition on the hematopoietic regulatory function of MSCs. C57BL/6NTaq mice were divided into control and protein malnutrition groups, which received, respectively, a normal protein diet (12% casein) and a low protein diet (2% casein). The results showed that protein malnutrition altered the synthesis of SCF, TFG-ß, Angpt-1, CXCL-12, and G-CSF by MSCs. Additionally, MSCs from the protein malnutrition group were not able to maintain the lymphoid, granulocytic and megakaryocytic-erythroid differentiation capacity compared to the MSCs of the control group. In this way, the comprehension of the role of MSCs on the regulation of the hematopoietic cells, in protein malnutrition states, is for the first time showed. Therefore, we infer that hematopoietic alterations caused by protein malnutrition are due to multifactorial alterations and, at least in part, the MSCs' contribution to hematological impairment.

Hemopoese em desnutrição proteica: caracterização do estroma medular e avaliação da participação do cálcio / Hematopoiesis in protein malnutrition: characterization of bone marrow stroma and evaluation of calcium participation

A desnutrição proteica continua sendo um dos principais problemas nutricionais do mundo. Trabalhos de nosso laboratório e de outros autores evidenciam que entre as alterações presentes na desnutrição proteica, está a alteração do tecido hemopoético, com modificações em componentes da matriz extracelular, alterações no ciclo celular da célula tronco/progenitora hemopoética, redução da produção de precursores hemopoéticos, tanto na série eritrocitária como na série leucocitária, levando a anemia e leucopenia. Os mecanismos de participação do Ca2+ nas células da medula óssea são pouco conhecidos, porém, sabe-se que ele atua no processo de hemopoese. Têm sido descrito que elevações da concentração de Ca2+ citoplasmático induzem a proliferação e diferenciação de células mielóides. A ação dessa via em indivíduos desnutridos também é pouco conhecida. Este estudo tem como objetivo avaliar o estabelecimento da celularidade medular in vitro, bem como investigar mecanismos moleculares envolvidos na proliferação e diferenciação dessa celularidade, além de avaliar a ação do cálcio na presença da interleucina-3 em células-tronco hemopoéticas murinas e sua modulação para avaliar alterações na via das MAPKs. Camundongos C57BL/6, machos e adultos foram submetidos à desnutrição proteica e, após a perda de aproximadamente 20% de seu peso corporal, as células da medula óssea foram colhidas. Essas células foram imunofenotipadas, além de reagirem com anticorpos específicos para caracterização da célula-tronco hemopoética e proteínas da via de sinalização de cálcio intracelular. Observamos que a celularidade do estroma medular em cultura de longa duração de animais desnutridos é alterada, principalmente em células de origem mesenquimal, que aparecem em maior número em desnutridos ao longo dos dias de cultura. Além disso, as ondas de cálcio intracelular estavam diminuídas em animais desnutridos, bem como as proteínas p-PKC, p-PLCy, CAMKII, p-AKT e p-STAT5 não respondem ao estímulo de IL-3, levando a uma deficiência da expressão das MAPK: ERK 1/2, JNK e p38. A desnutrição proteica pode causar alterações na celularidade estromal da medula óssea e na diferenciação das células tronco hemopoéticas pela via das MAPKs estimulada por IL-3

Protein malnutrition remains one of the world's major nutritional problems. Studies from our laboratory and others shown that alterations in protein malnutrition include hemopoietic tissue alterations, changes in extracellular matrix components, changes in the hemopoietic stem/progenitor cell tissue, reduction in the production of hemopoietic precursors, in the erythroid series as in the mieloyd series, leading to anemia and leukopenia. Mechanisms of Ca2+ participation in bone marrow cells are poorly understood, but no hemopoiesis has been developed. Elevations of cytoplasmic Ca2+ concentration in proliferation and differentiation of myeloid cells were included. Such an action through malnourished animals is also a little known. This study aims to evaluate the establishment of cellularity in vitro as well as investigate the molecular involvement in cell proliferation and differentiation, as well as to evaluate the action of calcium in the presence of IL-3 in hemopoietic stem cells and its modulation by analytical evaluations in the MAPKs pathway. C57BL/6, male adult mices were subjected to protein restriction and, after loss of approximately 20% of their body weight, bone marrow cells were harvested. These were immunophenotyped in addition to specific activation terms for the hemopoietic stem cell and intracellular signaling pathway proteins. We observed that the bone marrow cells in long-term culture of malnourished animals is altered, mainly in cells of mesenchymal origin, which appears in greater numbers in undernourished throughout the days of culture. In addition, as intracellular calcium waves decreased in malnourished animals, as well as the p-PKC, p-PLC, CAMKII, p-AKT and p-STAT5 proteins did not respond to IL-3, sugesting expression of the expression of MAPK: ERK 1/2, JNK and p38. Protein malnutrition may have changes in bone marrow capacity and differentiation of hemopoietic stem cells through IL-3-stimulated MAPKs

The effects of protein malnutrition on the TNF-RI and NF-κB expression via the TNF-α signaling pathway.

Malnutrition is a nutritional condition that can affect many aspects of the immunological response, including by decreasing cell migration and stimulating phagocytosis; the bactericidal response; changes in reactive oxygen and nitrogen species production; and the production of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-α). This cytokine is primarily produced by macrophages and is associated with a wide range of biological activities, including inflammatory processes, growth, differentiation, and apoptosis. TNF-α acts through the activation of TNF receptors, and mainly receptor I (TNF-RI), which is responsible for most of the effects of TNF-α. This activation triggers a series of intracellular events that result in the activation of the transcription factor NF-κB. In this study, we evaluated the expression of the transcription factor NF-κB, mediated by TNF-α through TNF-RI, in a protein malnutrition (PM) model. Adult male BALB/c mice were submitted to PM, and after loss of approximately 20% of their body weight, their peritoneal macrophages were collected and cultivated with or without TNF-α. The expression of TNF-RI and proteins in its signaling pathway (TRADD, TRAF, RIP, IKK, IKB-α, pIKB-α, NF-κB, and pNF-κB) were evaluated, as well as cytokine production (IL-1α, IL-1ß, IL-6, and IL-12). The compiled results highlight that the malnourished animals presented anemia, leukopenia, and decreased peritoneal cellularity. TNF-RI expression was reduced in the malnourished animals, and NF-κB phosphorylation was also reduced, in association with reduced production of IL-1ß and IL-12. In this study, we observed aspects related to the innate immune response, and the outcome data allowed us to conclude that nutritional status interferes with the macrophage activation and the response capabilities of these cells.

Effect of high-fat diet upon inflammatory markers and aortic stiffening in mice.

Changes in lifestyle such as increase in high-fat food consumption are an important cause for vascular diseases. The present study aimed to investigate the involvement of ACE and TGF- ß in the aorta stiffness induced by high-fat diet. C57BL/6 male mice were divided in two groups according to their diet for 8 weeks: standard diet (ST) and high-fat diet (HF). At the end of the protocol, body weight gain, adipose tissue content, serum lipids and glucose levels, and aorta morphometric and biochemical measurements were performed. Analysis of collagen fibers by picrosirius staining of aorta slices showed that HF diet promoted increase of thin (55%) and thick (100%) collagen fibers deposition and concomitant disorganization of these fibers orientations in the aorta vascular wall (50%). To unravel the mechanism involved, myeloperoxidase (MPO) and angiotensin I converting enzyme (ACE) were evaluated by protein expression and enzyme activity. HF diet increased MPO (90%) and ACE (28%) activities, as well as protein expression of ACE. TGF-ß was also increased in aorta tissue of HF diet mice after 8 weeks. Altogether, we have observed that the HF diet-induced aortic stiffening may be associated with increased oxidative stress damage and activation of the RAS in vascular tissue.