Amanda Nogueira-Pedro

Nitric Oxide‐Induced Murine Hematopoietic Stem Cell Fate Involves Multiple Signaling Proteins, Gene Expression, and Redox Modulation

There are a growing number of reports showing the influence of redox modulation in cellular signaling. Although the regulation of hematopoiesis by reactive oxygen species (ROS) and reactive nitrogen species (RNS) has been described, their direct participation in the differentiation of hematopoietic stem cells (HSCs) remains unclear. In this work, the direct role of nitric oxide (NO•), a RNS, in the modulation of hematopoiesis was investigated using two sources of NO•, one produced by endothelial cells stimulated with carbachol in vitro and another using the NO•‐donor S‐nitroso‐N‐acetyl‐d,l‐penicillamine (SNAP) in vivo. Two main NO• effects were observed: proliferation of HSCs—especially of the short‐term HSCs—and its commitment and terminal differentiation to the myeloid lineage. NO•‐induced proliferation was characterized by the increase in the number of cycling HSCs and hematopoietic progenitor cells positive to BrdU and Ki‐67, upregulation of Notch‐1, Cx43, PECAM‐1, CaR, ERK1/2, Akt, p38, PKC, and c‐Myc. NO•‐induced HSCs differentiation was characterized by the increase in granulocytic‐macrophage progenitors, granulocyte–macrophage colony forming units, mature myeloid cells, upregulation of PU.1, and C/EBPα genes concomitantly to the downregulation of GATA‐3 and Ikz‐3 genes, activation of Stat5 and downregulation of the other analyzed proteins mentioned above. Also, redox status modulation differed between proliferation and differentiation responses, which is likely associated with the transition of the proliferative to differentiation status. Our findings provide evidence of the role of NO• in inducing HSCs proliferation and myeloid differentiation involving multiple signaling. Stem Cells 2014;32:2949–2960

A high-fat diet increases interleukin-3 and granulocyte colony-stimulating factor production by bone marrow cells and triggers bone marrow hyperplasia and neutrophilia in wistar rats

It is well established that the excessive consumption of a high-fat diet (HFD) results in overweight, obesity and an increase in leptin concentrations, which triggers a chronic inflammatory condition that is associated with a high white blood cell count. Two-month-old male Wistar rats were fed a control (CON) diet or an HFD for 12 weeks. After this period, hemogram, myelogram and biochemical parameters were evaluated along with the cell cycle and the percentage of CD34+ cells in the bone marrow as well as cell proliferation and differentiation assays and the production of stem cell factor, interleukin 3 (IL-3), granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage colony-stimulating factor (GM-CSF). The HFD animals exhibited leukocytosis and neutrophilia with increased C-reactive protein, leptin, cholesterol and triglyceride concentrations. In the HFD group, the bone marrow revealed myeloid hyperplasia, especially of the granulocytic compartment with a higher percentage of CD34+ cells and a higher percentage of cells in the G2/S/M cell cycle phases. In addition, the HFD bone marrow cells had a higher capacity to proliferate and differentiate into granulocytic cells in an in vitro system and a higher capacity to produce IL-3 and G-CSF. These data led us to infer that the HFD induces leukocytosis and neutrophilia suggesting alterations in hematopoiesis system modulation.

α‐Tocopherol induces hematopoietic stem/progenitor cell expansion and ERK1/2‐mediated differentiation

Tocopherols promote or inhibit growth in different cell types. In the hematopoietic system, the radioprotective property of tocopherols is thought to act through the expansion of primitive hematopoietic cells. However, the mechanisms activated by tocopherols and which HPs are affected remain poorly understood. To better address these questions, mice were treated with α‐tocopherol, and its effects were investigated in the BM microenvironment. α‐Tocopherol induced increased proliferation in HSC/HP cells, leading to BM hyperplasia. In addition, differentiation to the granulocytic/monocytic lineage was enhanced by α‐tocopherol treatment. α‐Tocopherol treatment resulted in decreased basal phosphorylation of ERK1/2, PKC, and STAT‐5 in HSC/HP cells. In contrast, α‐tocopherol enhanced ERK1/2 activation in response to IL‐3 stimulation in HSC/HP cells without altering the expression of IL‐3Rs. Moreover, α‐tocopherol‐induced differentiation and ERK1/2 activation were abolished in mice pretreated with a MEK inhibitor (PD98059); however, pretreatment with PD98059 did not reduce the α‐tocopherol‐mediated increase in HSC/HP cells but instead, further enhanced their proliferation. Therefore, α‐tocopherol induces expansion of HSC/HP cells by a nonidentified intracellular pathway and granulocytic/monocytic differentiation through ERK1/2 activation.

The impaired viability of prostate cancer cell lines by the recombinant plant kallikrein inhibitor

Background: Kallikreins play a pivotal role in establishing prostate cancer. Results: In contrast to the classical Kunitz plant inhibitor SbTI, the recombinant kallikrein inhibitor (rBbKIm) led to prostate cancer cell death, whereas fibroblast viability was not affected. Conclusion: rBbKIm shows selective cytotoxic effect and angiogenesis inhibition against prostate cancer cells. Significance: New actions of rBbKIm may contribute to understanding the mechanisms of prostate cancer. Prostate cancer is the most common type of cancer, and kallikreins play an important role in the establishment of this disease. rBbKIm is the recombinant Bauhinia bauhinioides kallikreins inhibitor that was modified to include the RGD/RGE motifs of the inhibitor BrTI from Bauhinia rufa. This work reports the effects of rBbKIm on DU145 and PC3 prostate cancer cell lines. rBbKIm inhibited the cell viability of DU145 and PC3 cells but did not affect the viability of fibroblasts. rBbKIm caused an arrest of the PC3 cell cycle at the G0/G1 and G2/M phases but did not affect the DU145 cell cycle, although rBbKIm triggers apoptosis and cytochrome c release into the cytosol of both cell types. The differences in caspase activation were observed because rBbKIm treatment promoted activation of caspase-3 in DU145 cells, whereas caspase-9 but not caspase-3 was activated in PC3 cells. Because angiogenesis is important to the development of a tumor, the effect of rBbKIm in this process was also analyzed, and an inhibition of 49% was observed in in vitro endothelial cell capillary-like tube network formation. In summary, we demonstrated that different properties of the protease inhibitor rBbKIm may be explored for investigating the androgen-independent prostate cancer cell lines PC3 and DU145.

Hematopoietic stem cell expansion caused by a synthetic fragment of leptin

Leptin is a cytokine that regulates food intake, energy expenditure and hematopoiesis. Based on the tridimensional structure of the human leptin molecule, six fragments have been synthesized, (Ac-Lep23-47-NH2, [LEP1]; Ac-Lep48-71-NH2, [LEP2]; Ac-Lep72-88-NH2, [LEP3]; Ac-Lep92-115-NH2, [LEP4], Ac-[Ser(117)]-Lep116-140-NH2, [LEP5] and Ac-Lep141-164-NH2, [LEP6]), and their effects on hematopoiesis were evaluated. The mice were treated with 1mg/kg LEP5 for 3 days. The mature and primitive hematopoietic populations were quantified. We observed that the mature populations from the bone marrow and spleen were not affected by LEP5. However, the peptide caused at least a two-fold increase in the number of hematopoietic stem cells, the most primitive population of the bone marrow. Additionally, the number of granulocyte/macrophage colony-forming units produced by bone marrow cells in methylcellulose also increased by 40% after treatment with LEP5, and the leptin receptor was activated. These results show that the leptin fragment LEP5 is a positive modulator of the in vivo expansion of hematopoietic stem cells.

Hydrogen peroxide (H2O2) induces leukemic but not normal hematopoietic cell death in a dose-dependent manner

Over the last few years, studies have suggested that oxidative stress plays a role in the regulation of hematopoietic cell homeostasis. In particular, the effects of hydrogen peroxide (H2O2) range from hematopoietic cell proliferation to cell death, depending on its concentration in the intracellular milieu. In this work, we evaluated the effects of an oxidative environment on normal and leukemic hematopoietic cells by stimulating normal human (umbilical cord blood) and murine (bone marrow) hematopoietic cells, as well as human myeloid leukemic cells (HL-60 lineage), upon H2O2 stimulus. Total cell populations and primitive subsets were evaluated for each cell type. H2O2 stimulus induces HL-60 cell death, whereas the viability of human and murine normal cells was not affected. The effects of H2O2 stimulus on hematopoietic stem/progenitor cell subsets were examined and the normal primitive cells were found to be unaffected; however, the percentage of leukemic stem cells (LSC) increased in response to H2O2, while clonogenic ability of these cells to generate myeloid clones was inhibited. In addition, H2O2 stimulus caused a decrease in the levels of p-AKT in HL-60 cells, which most likely mediates the observed decrease of viability. In summary, we found that at low concentrations, H2O2 preferentially affects both the LSC subset and total HL-60 cells without damage normal cells.

Gomesin acts in the immune system and promotes myeloid differentiation and monocyte/macrophage activation in mouse

Due to the cytotoxic effect of antimicrobial peptides (AMP) against several microorganism and tumor cells has been proposed their association with the immune system. However, just a few reports have shown this relationship. In this study, mice were treated with gomesin, a β-hairpin AMP that exhibit high cytotoxicity against bacterial and tumor cells. Different effects in the immune system were observed, such as, decrease of CD3+ in T lymphocytes (Control: 17.7±1.4%; Gomesin: 7.67±1.2%) and in hematopoietic progenitors and increase of hematopoietic stem cell (Control: 0.046±0.004%; Gomesin: 0.067±0.003%), B220+ B lymphocytes (Control: 38.63±1.5%; Gomesin: 47.83±0.48%), and Mac-1+F4/80+ macrophages (Control: 11.76±3.4%; Gomesin: 27.13±4.0%). Additionally, macrophage increase was accompanied by an increase of macrophage phagocytosis (Control 20.85±1.53; Gomesin 31.32±1 Geometric mean), interleukin 6 (Control: 47.24±1.9ng/mL; Gomesin: 138.68±33.68ng/mL) and monocyte chemoattractant protein-1 (Control: 0.872±0.093ng/mL; Gomesin: 1.83±0.067ng/mL). Thus, this report showed immunomodulatory activity of gomesin in the immune system of mice.

Effects of Sleep Deprivation on Mice Bone Marrow and Spleen B Lymphopoiesis

B lymphocytes are immune cells crucial for the maintenance and viability of the humoral response. Sleep is an essential event for the maintenance and integrity of all systems, including the immune system (IS). Thus, sleep deprivation (SD) causes problems in metabolism and homeostasis in many cell systems, including the IS. In this study, our goal was to determine changes in B lymphocytes from the bone marrow (BM) and spleen after SD. Three‐month‐old male Swiss mice were used. These mice were sleep deprived through the modified multiple platform method for different periods (24, 48, and 72 h), whereas another group was allowed to sleep for 24 h after 72 h of SD (rebound group) and a third group was allowed to sleep normally during the entire experiment. After this, the spleen and BM were collected, and cell analyses were performed. The numbers of B lymphocytes in the BM and spleen were reduced by SD. Additionally, reductions in the percentage of lymphocyte progenitors and their ability to form colonies were observed. Moreover, an increase in the death of B lymphocytes from the BM and spleen was associated with an increase in oxidative stress indicators, such as DCFH‐DA, CAT, and mitochondrial SOD. Rebound was not able to reverse most of the alterations elicited by SD. The reduction in B lymphocytes and their progenitors by cell death, with a concomitant increase in oxidative stress, showed that SD promoted a failure in B lymphopoiesis. J. Cell. Physiol. 231: 1313–1320, 2016.

A Synthetic Fragment of Leptin Increase Hematopoietic Stem Cell Population and Improve Its Engraftment Ability

Several studies have shown the important actions of cytokine leptin that regulates food intake and energy expenditure. Additionally, the ability to modulate hematopoiesis has also been demonstrated. Previous reports have shown that some synthetic sequences of leptin molecules can activate leptin receptor. Herein, decapeptides encompassing amino acids from positions 98 to 122 of the leptin molecule were constructed to evaluate their effects on hematopoiesis. Among them, the synthetic peptide Lep110‐119‐NH2 (LEP F) was the only peptide that possessed the ability to increase the percentage of hematopoietic stem cells (HSC). Moreover, LEP F also produced an increase of granulocyte/macrophage colony‐forming units and activated leptin receptor. Furthermore, LEP F also improves the grafting of HSC in bone marrow, but did not accelerate the recovery of bone marrow after ablation with 5‐fluorouracil. These results show that LEP F is a positive modulator of the in vivo expansion of HSC and could be useful in bone marrow transplantation. J. Cell. Biochem. 116: 1334–1340, 2015.

Leptin Fragment Increase Hematopoietic Stem Cell Population and Improve its Engraftment Ability

Several studies have shown the important actions of cytokine leptin that regulates food intake and energy expenditure. Additionally, the ability to modulate hematopoiesis has also been demonstrated increasing hematopoietic progenitors. Previous reports of our group have shown that some synthetic sequences of leptin molecules can activate leptin receptor [1-3]. Herein, decapeptides encompassing amino acids from positions 98 to 122 of the leptin molecule were constructed to evaluate their effects on hematopoiesis [4].