Bone Marrow Proliferation Research Articles

Cyclophosphamide induces bone marrow to yield higher numbers of precursor dendritic cells in vitro capable of functional antigen presentation to T cells in vivo

We have shown recently that cyclophosphamide (CTX) treatment induced a marked increase in the numbers of immature dendritic cells (DCs) in blood, coinciding with enhanced antigen-specific responses of the adoptively transferred CD8 + T cells. Because this DC expansion was preceded by DC proliferation in bone marrow (BM), we tested whether BM post CTX treatment can generate higher numbers of functional DCs. BM was harvested three days after treatment of C57BL/6 mice with PBS or CTX and cultured with GM-CSF/IL-4 in vitro. Compared with control, BM from CTX-treated mice showed faster generation and yielded higher numbers of DCs with superior activation in response to toll-like receptor (TLR) agonists. Vaccination with peptide-pulsed DCs generated from BM from CTX-treated mice induced comparable adjuvant effects to those induced by control DCs. Taken together, post CTX BM harbors higher numbers of DC precursors capable of differentiating into functional DCs, which be targeted to create host microenvironment riches in activated DCs upon treatment with TLR agonists.

Anaplasma phagocytophilum and Ehrlichia muris induce cytopenias and global defects in hematopoiesis

Peripheral blood cytopenias result from a wide variety of infectious diseases and may significantly complicate the prognosis and outcome of those diseases. Infection with Gram-negative, LPS-negative members of the Anaplasmataceae family, including Anaplasma phagocytophilum and Ehrlichia muris, results in bi- or pancytopenia. The specific mechanisms responsible for the cytopenias are unknown. Viruses and bacteria have profound yet often discrepant effects on bone marrow (BM) and circulating numbers of blood cells. Multiple line-age cytopenias (bi- and pancytopenia) are a frequent sequel of viral infections [1]. Different viruses can directly infect BM stromal cells or haematopoietic stem cells and/or modulate cytokine production by accessory cells. Infections with Gram-negative, LPS-positive bacteria result in an endotoxin-mediated, acute phase, proinflammatory cascade of events that result in granulocyte efflux from the BM, peripheral neutrophilia and BM hyperplasia [2]. In contrast, gram-negative, LPS-negative bacteria, including members of the Anaplasmataceae family, elicit a very different, poorly-characterised, cascade of events. Haematologic alterations in both natural and experimental infection include a marked thrombocytopenia, moderate leukopenia (lymphopenia and neutropenia) and mild to moderate, non-regenerative anaemia [3,4]. Cytopenias vary from profound and irreversible (for example, dogs with chronic E. canis infection) to moderate and transient. Paradoxically, although Anaplasma family organisms display cell tropism for specific haematopoietic cell lineages, infection, regardless of aetiologic agent, host species or cell target, is characterised by bicytopenia or, occasionally, pancytopenia. This suggests the potential for more global haematopoietic disturbances. We hypothesised that the cytopenias may be due in part to bacteria-induced haematopoietic suppression. C57/Bl6 mice were infected intraperitoneally with E. muris and samples were collected on days 0–60 post-infection. C3H/HeN mice were infested with A. phagocytophilum-infected Ixodes scapularis nymphal ticks and sampled on days 0–21 post-infection. Blood samples were collected for a complete blood cell analysis and quantitative PCR for pathogen detection (A. phagocytophilum p44 DNA). BM was collected for haematopoietic colony-forming assays, cytology, histology and quantitative PCR. Cultured BM supernatants from A. phagocytophilum infected mice were collected for cytokine analysis. Thrombocytopenia and anaemia (bicytopenia) developed in both E. muris and A. phagocytophilum-infected mice. Leukopenia was variable. A. phagocytophilum infection induced significant decreases in BM proliferation and differentiation from day 4 to day 21 post-infection, concurrent with the cytopenias (Fig. 1). E. muris infection resulted in a similarly dramatic decrease in BM proliferation and differentiation on days 8 to 14 post-infection. Decreases in BM haematopoietic production included all lineages. Haematopoietic colony forming assays are supplemented with cytokines and assess haematopoietic stem cell/progenitor cell activity in the absence of stromal cells. As such, findings support infection-induced alterations in stem cell differentiation, proliferation or mobilisation. Both thrombocytopenia and myelosuppression were more severe in mice infected with A. phagocytophilum via tick bite compared with mice infected intraperitoneally in previous studies. Thrombocytopenia did not correlate with pathogen burden in blood, as assessed by quantitative PCR, but was strongly correlated with BM colony numbers in A. phagocytophilum-infected mice. This temporal association suggests that rapid changes in BM may be contributing to alterations in circulating platelet numbers. Cytologic evaluation of BM revealed infection-induced lymphocyte depletion and a reciprocal granulocytic hyperplasia in both infection models, similar to kinetics noted in other models of inflammation [5]. In addition, A. phagocytophilum infection resulted in BM production of myelosuppressive chemokines, including the murine interleukin-8 homologues, macrophage inflammatory protein-2 and KC. Findings are compatible with previous work that demonstrated a predominance of myelosuppressive chemokines in human BM after A. phagocytophilum infection [6]. Anaplasma family pathogens appear to perturb normal haematopoiesis, resulting in shifts in BM cell populations and transient failure of haematopoietic colony formation, which may contribute to peripheral blood cytopenias. Fig. 1 Bone marrow was collected from Anaplasma phagocytophilum-infected and uninfected mice on days 0–21 after tick-borne infection. An aliquot of BM was plated in duplicate and cultured in complete MethoCult media for 7–10 days (StemCell Technologies) ...

Identification of MicroRNA Expression Patterns and Definition of a MicroRNAs/mRNA Regulatory Network in Distinct Molecular Groups of Multiple Myeloma.

Abstract 2824Poster Board II-800Multiple myeloma (MM) is a malignant proliferation of bone marrow (BM) plasma cells (PCs), characterized by a profound genomic instability involving both numerical and structural chromosomal aberrations of potential prognostic relevance. The discovery of microRNA (miRNA) genes, encoding for a class of small non-coding RNAs involved in the regulation of cell cycle, survival and differentiation programs, has added a further level of complexity to normal and cancer cell biology; it has been suggested that chromosomal abnormalities and other types of genetic or epigenetic alterations might contribute to miRNA deregulation in cancer. To date, only little evidence of miRNA expression/deregulation in multiple myeloma (MM) has been reported. To characterize miRNA expression profiling of MM plasma cells (PCs) and integrate miRNA expression data with other molecular features of MM patients, global miRNA expression profiles of PCs isolated from BM biopsies of 38 newly diagnosed MM, 2 plasma cell leukemia patients, and 3 healthy donors were generated using the Agilent Human miRNA microarray V2 (representing 723 human mature miRNAs from the Sanger miRBase v10.1). All of the patients had previously been characterized by FISH for the main IGH translocations and other genetic abnormalities; they were also profiled for global gene expression by means of Affymetrix U133A arrays; nineteen of the patients profiled for miRNA expression underwent genome-wide DNA analysis using Affymetrix GeneChip® Human Mapping 50K XbaI microarrays. An unsupervised analysis of the samples, using conventional hierarchical clustering algorithm and based on the most variably expressed miRNAs across the dataset, grouped the PCs from healthy donors separately from MM PCs; among the pathological samples, the most striking finding was that the seven patients with t(4;14) (TC4) were tightly clustered, as were four out of the five samples with translocated MAF genes (TC5). A partial grouping of the TC2 cases (mostly hyperdiploid) was also observed, whereas the TC1, showing t(11;14), and TC3 (mostly expressing Cyclin D2), samples were dispersed along the dendrogram. A multiclass supervised analysis of the miRNA expression between the members of the 5 TC groups highlighted specific miRNA signatures, in particular characterizing the TC4 and TC5 groups. A slight miRNA signature was observed in t(11;14) cases and TC2 group, whereas we could not identify any TC3-specific miRNA signature. None of the differentially expressed miRNAs in patients with specific IGH translocations maps to the rearranged chromosomal regions. The levels of some differentially expressed miRNAs were quantified by means of Q-RT-PCR in all of the 43 samples, using specific TaqMan® microRNA assays (Applied Biosystems); a linear correlation analysis indicated very good correspondence between microarrays and Q-RT-PCR data. Furthermore, the expression of specific miRNAs was also evaluated in 14 additional cases, four carrying t(11;14), seven t(4;14), and three t(14;16) or t(14;20): as well, in this broaden panel the selected miRNAs confirmed their TC-specific over-expression. The occurrence of other lesions, such as 1q gain, 13q and 17p deletions, and hyperdiploidy, was slightly characterized by specific miRNA signatures. Furthermore, the integrated analysis with the genomic profiles revealed the occurrence of several allelic imbalances significantly associated with the altered expression of miRNAs located in the involved regions, such as let-7b at 22q13.31 (loss) and miR-142-3p at 17q22 (gain), or miR-140-3p at 16q22 (loss-of-heterozygosity). Finally, in an attempt to define the consequences of deregulated miRNA expression, we performed an integrative analysis based on computational target prediction, miRNA and mRNA profiling. Specifically, we searched for putative functional targeting relationships in MM cells supported by expression data, i.e. anti-correlations between miRNA and target mRNA expression profiles, and thus defined a global miRNAs/mRNAs regulatory network. Our data provide the first evidence of miRNA deregulation in the context of the molecular subtypes of MM, and represent the first attempt to define the complex of miRNAs/mRNAs regulatory relationships, aimed at deepening our understanding of the involvement of specific miRNAs and target genes in the pathology of the disease. Disclosures:No relevant conflicts of interest to declare.

Abstract 5524: Progenitor Cell Mobilization is FGF-2 Dependant and Results in the Activation of Osteoclasts

Background Fibroblast growth factor-2 (FGF2) regulates proliferation of bone marrow (BM) stromal cells and has pro-angiogenic effects regulating endothelial cell growth, migration, and reendothelialization. Here we determine the underlying molecular mechanisms of FGF2-dependent statin-mediated progenitor cell mobilization. Methods and Results To determine the molecular mechanisms of FGF2-mediated progenitor cell mobilization, FGF2−/− and wild type (wt) mice were treated with atorvastatin (10mg/kg body weight s.c. per day) and placebo. In contrast to wt mice, the number of Sca1/flk-1 positive EPC in peripheral blood (flow cytometry) of atorvastatin treated FGF2−/− mice did not increase. This was due to an accumulation of Sca1/flk-1 positive cells within the BM. Inhibition of EPC mobilization in FGF2−/− mice was associated with a delay in reendothelialization in a mouse model of a focal endothelial cell denudation and a severe reduction in neoangiogenesis despite statin treatment. BM transplantation experiments demonstrated that reconstitution of FGF2−/− mice with wt stem cells completely restored statin-mediated EPC mobilization while wt mice reconstituted with FGF2−/− stem cells developed a severe mobilization defect associated with an impaired endothelial dependent vasorelaxation (organ chamber) and enhanced neointima formation. In wt mice, statin treatment was associated with an increased number of activated osteoclasts compared to placebo treated mice which are known to play a pivotal role in stem cell mobilization. Treatment with osteoclast-activating RANKL induced an effective mobilization of Sca1/flk1 and Sca1/CD117 positive progenitor cells and enhanced the number of early outgrowth EPC. Conclusion Medullary FGF2 is essential in the statin-mediated mobilization of EPC from BM into peripheral blood. A reduction in the number and activation status of osteoclasts may account for the impairment in EPC mobilization.

Production of biologically active human myelocytokines in plants

An effective system for expression of human granulocyte and granulocyte macrophage colony-stimulating factors (hG-CSF and hGM-CSF) in Nicotiana benthamiana plants was developed using viral vector based on tobacco mosaic virus infecting cruciferous plants. The genes of target proteins were cloned into the viral vector driven by actin promoter of Arabidopsis thaliana. The expression vectors were delivered into plant cells by agroinjection. Maximal synthesis rate was detected 5 days after injection and was up to 500 and 300 mg per kg of fresh leaves for hG-CSF and hGM-CSF, respectively. The yield of purified hG-CSF and hGM-CSF was 100 and 50 mg/kg of fresh leaves, respectively. Recombinant plant-made hG-CSF and hGM-CSF stimulated proliferation of murine bone marrow and human erythroleucosis TF-1 cells, respectively, at the same rate as the commercial drugs.

Using Molecular Imaging to Quantitatively Estimate the Distribution of Proliferating Bone Marrow in Adult Cancer Patients

Using Molecular Imaging to Quantitatively Estimate the Distribution of Proliferating Bone Marrow in Adult Cancer Patients

SAPHO syndrome with adrenal deficiency: a case report.

The SAPHO syndrome (synovitis, acne, pustulosis, hyperostosis, osteomyelitis) is a rare painful disorder, usually with a good long-term prognosis. Its etiology remains unclear, and various treatment regimens frequently fail to control the disease. A 46-year-old Caucasian female was referred for anterior chest wall and back pain. Physical examination was unremarkable except for skin lesions noted on soles of both feet, extremities and the face. A thoracic magnetic resonance imaging study demonstrated a lesion characterized with bone marrow edema and proliferation of soft tissue in the sternum. A brain MRI was requested secondary to the elevated prolactin level which was compatible with empty sella syndrome. The case presented here has the unique feature of adrenal deficiency presenting alongside the SAPHO syndrome and is presented as the first case reported. This syndrome could become complicated with different organ system involvement other than bone and skin. There is a need further studies that will explore the weak relationship between SAPHO syndrome and adrenal deficiency.

Effects of Imatinib Mesylate on Renin–Angiotensin System (RAS) Activity during the Clinical Course of Chronic Myeloid Leukaemia

The renin-angiotensin system (RAS) is involved in cell growth, proliferation and differentiation in bone marrow in an autocrine-paracrine manner, and it modulates normal and neoplastic haematopoietic cell proliferation. This study aimed to assess expressions of the RAS components, renin, angiotensinogen and angiotensin-converting enzyme (ACE), during imatinib mesylate treatment of patients with chronic myeloid leukaemia (CML). Expressions of RAS components were studied in patients with CML at the time of diagnosis (n = 83) and at 3, 6 and 12 months after diagnosis (n = 35) by quantitative real-time polymerase chain reaction. De novo CML patients had increased ACE, angiotensinogen and renin mRNA levels and these expression levels decreased following administration of imatinib. The RAS activities were significantly different among Sokal risk groups of CML, highlighting the altered biological activity of RAS in neoplastic disorders. The results of this study confirm that haematopoietic RAS affects neoplastic cell production, which may be altered via administration of tyrosine kinase inhibitors such as imatinib mesylate.

Treatment of severe aplastic anemia by immunosuppressor anti-lymphocyte globulin/anti-thymus globulin as the chief medicine in combination with Chinese drugs

To study the therapeutic effect of combined therapy with Chinese drugs and immuno-suppressors, mainly anti-lymphocyte globulin/anti-thymus globulin (ALG/ATG), for the treatment of severe aplastic anemia (SAA), the efficacy associated factors and adverse effects as well. A retrospective analysis was conducted on 65 patients with SAA treated by combined therapy which was supplemented with cyclosporin A, androgen, hematopoietic growth factor, etc. Of the 57 patients followed-up, 26 (45.6%) were basically cured, 15 (26.3%) remitted, and 8 (14.0%) improved markedly, the total effective rate being 85.9%. By separately comparing with a single item of clinical data, it was shown that the therapeutic effectiveness was correlated, to a certain extent, with age, illness duration, neutrophil count, and bone marrow proliferation in patients before treatment, as well as with infection that occurred in the follow-up period. It was obviously higher in patients with peripheral neutrophil count > 0.5 x 10 10(9)/L (P<0.05). Various degrees of serum sickness-like reactions occurred in the treatment of 36 patients, including fever in 36 (63.2%), skin rash in 8 (14.0%), and musculoskeletal pain in 5 (8.8%). The therapeutic effect of combined therapy with Chinese drugs and ALG/ATG in treating SAA could be affirmed, showing some superiority as compared with Western medicine alone. The patients' age, duration of illness, neutrophil count, and bone marrow proliferation before treatment, and degree of infection that occurred could affect the therapeutic efficacy to a certain extent. Adverse reactions resulting from the combined therapy are less, showing the toxicity reducing and effect enhancing action of Chinese drugs.

Cell adhesion and proliferation evaluation of SFF-based biodegradable scaffolds fabricated using a multi-head deposition system

Scaffolds composed of biodegradable polymers and biocompatible ceramics are being used as substitutes for tissue engineering. In the development of such techniques, scaffolds with a controllable pore size and porosity were manufactured using solid free-form fabrication (SFF) methods to investigate the effects of cell interactions such as cell proliferation and differentiation. In this study, we describe the adhesion of human bone marrow stromal cells (hBMSCs) and proliferation characteristics of various scaffolds, which consist of biodegradable materials, fabricated using a multi-head deposition system (MHDS) that we developed. The MHDS uses novel technology that enables the production of three-dimensional (3D) microstructures. Fabrication of 3D tissue engineering scaffolds using the MHDS requires the combination of several technologies, such as motion control, thermal control, pneumatic control and computer-aided design/computer-assisted manufacturing software. The effects of a polymer and ceramic on a tissue scaffold were evaluated through mechanical testing and cell interaction analysis of various kinds of scaffolds fabricated using polycaprolactone, poly-lactic-co-glycolic acid and tri-calcium phosphate for bone regeneration applications. Based on these results, the feasibility of application to the tissue engineering of SFF-based 3D scaffolds fabricated by the MHDS was demonstrated.

Effects of continuous and pulsatile PTH treatments on rat bone marrow stromal cells

Bone marrow stromal cells (MSCs) differentiation and proliferation are controlled by numerous growth factors and hormones. Continuous parathyroid hormone (PTH) treatment has been shown to decrease osteoblast differentiation, whereas pulsatile PTH increases osteoblast differentiation. However, the effects of PTH treatments on MSCs have not been investigated. This study showed continuous PTH treatment in the presence of dexamethasone (DEX) promoted osteogenic differentiation of rat MSCs in vitro, as demonstrated by increased alkaline phosphatase (ALP) activity, number of ALP expressing cells, and up-regulation of PTH receptor-1, ALP, and osteocalcin mRNA expressions. In contrast, pulsatile PTH treatment was found to suppress osteogenesis of rat MSCs, possibly by promoting the maintenance of undifferentiated cells. Additionally, the observed effects of PTH were strongly dependent on the presence of DEX. MSC proliferation however was not influenced by PTH independent of treatment regimen and presence or absence of DEX. Furthermore, our work raised the possibility that PTH treatment may modulate stem/progenitor cell activity within MSC cultures.

Mechanical force promotes proliferation and early differentiation of bone marrow derived osteoblast-like cells in vitro

The aim of this study was to examine the effect of centrifugal force on rat bone marrow osteoblast-like cells (RBM) in vitro. RBM were seeded and cultured for 2 days. Centrifugal forces were added to the cells in the culture dishes using a centrifuge for 20 min either at 600 rpm or 4,800 rpm. The cells without centrifugation were used as control. The expression of HSP27 mRNA of both experimental groups after addition of centrifugal force for 3 hrs was significantly up-regulated compared with the control group. In the 4,800 rpm groups, the cells showed contracted shapes compared to the controls and the 600 rpm groups after treatment with centrifugal force. The maximum height of each cell in the control group was 2.00 ± 0.33 μm. The maximum height of each cell in the 600 rpm group was 1.50 ± 0.23 μm, and in the 4,800 rpm group it was 1.13 ± 0.23 μm. The proliferation ratio in the 4,800 rpm group at day 3 was significantly higher than in the other group. The expression of type I collagen and alkaline phosphatase (ALP) mRNAs in both experimental groups increased at earlier time periods compared to the control groups. ALP activity revealed no significant difference between the experimental groups and the control group. A centrifugal force of 4,800 rpm might promote the proliferation and early differentiation of rat bone marrow cells.

SAPHO syndrome with adrenal deficiency: a case report

IntroductionThe SAPHO syndrome (synovitis, acne, pustulosis, hyperostosis, osteomyelitis) is a rare painful disorder, usually with a good long-term prognosis. Its etiology remains unclear, and various treatment regimens frequently fail to control the disease.Case presentationA 46-year-old Caucasian female was referred for anterior chest wall and back pain. Physical examination was unremarkable except for skin lesions noted on soles of both feet, extremities and the face. A thoracic magnetic resonance imaging study demonstrated a lesion characterized with bone marrow edema and proliferation of soft tissue in the sternum. A brain MRI was requested secondary to the elevated prolactin level which was compatible with empty sella syndrome.ConclusionThe case presented here has the unique feature of adrenal deficiency presenting alongside the SAPHO syndrome and is presented as the first case reported. This syndrome could become complicated with different organ system involvement other than bone and skin. There is a need further studies that will explore the weak relationship between SAPHO syndrome and adrenal deficiency.

Effect of partial oxygen pressure on survival, proliferation, and differentiation of mouse bone marrow mesenchymal stem cells

Effects of partial oxygen pressure in a gas mixture surrounding the culture medium on survival, proliferation and differentiation of mesenchymal stem cells (MSC) isolated from mouse bone marrow was studied. It was found that 3% oxygen increased the survival of cells seeded at low density; the rate and duration of the MSC proliferation were also elevated. Effect of oxygen concentration on replicative activity of MSC was manifested as early as the first few days after the onset of cell growth. Studies of colony formation revealed that preincubation of cells with 21% oxygen had a negative effect on cell growth under 3% oxygen, while preincubation with 3% oxygen stimulated MSC proliferation in the presence of 21% oxygen. Notwithstanding, this effect of oxygen cannot be interpreted as unequivocally deleterious. Low partial oxygen pressure inhibited osteogenic differentiation of MSC, but adipogenic differentiation was insensitive to oxygen concentration. It is concluded that proliferation and differentiation of MSC depend critically on oxygen content in the culture medium.

Evidence for An Anti-Cancer Barrier in a Mixed Lineage Leukemia Mouse Model in Vivo.

Mixed Lineage Leukemia (MLL) mutations identify a unique group of acute leukemias with distinct biological and clinical features. Although the role of MLL in leukemogenesis has been extensively studied, a precise mechanism regarding the leukemogenic potential of MLL mutations is not known. We generated a switchable MLL-ENL-ERtm mouse model, in which the MLL-ENL oncogene has been introduced by homologous recombination and is controlled by the endogenous MLL promoter, thus, expressed at physiological levels. Due to fusion with the estrogen receptor ligand binding domain (ERtm), the MLL-ENL-ERtm protein activity is dependent on continuous provision of tamoxifen or 4-hydroxytamoxifen. The MLL-ENL-ERtm mice have developed a myeloproliferative disorder (MPD) characterized by persistent mature neutrophilia after 484,5 +/− 75,68 days of latency on a tamoxifen diet, in association with high white cell counts in peripheral blood, splenomegaly and occasionally with anemia. Blood smears showed large numbers of mature myeloid elements consisting of 40–80% neutrophils (non-segmented forms in abundance), admixed with immature myeloid elements, 3–11% monocytes and 2–6% myeloblasts. The phenotype of MPD also involved myelomonocytic proliferation with 35% immature monocytic cells in one animal and severe anemia with increased numbers of immature erythroid cells in peripheral blood in another animal. Hematoxylin- and eosin-stained sections of the bone marrow from MLL-ENL-ERtm mice revealed expansion of myeloid cell population with no signs of progressive dysplasia. We observed massive infiltration of myeloid cells (positive for myeloperoxidase) into spleen with various degree of loss of normal splenic architecture depending on disease progression. FACS profiles of both bone marrow and spleen cells showed a typical pattern of granulocyte/macrophage/monocyte surface marker expression (CD34-CD43+Mac- 1+Gr-1+CD16/32+). In vitro evaluation of hematopoetic progenitors derived from bone marrow of leukemic mice at the terminal stage of the disease revealed decreased numbers of BFU-Es and increased numbers of CFU-GMs and CFU-Gs compared to matched controls. These results correlated with the expansion of the myelomonocytic and reduction of the erythroid compartment observed in the bone marrow of these animals. The average size (cellularity) of the mutant myeloid colonies was much smaller than the colonies derived from the wild-type controls, which could be caused by a partial block of terminal differentiation of myeloid progenitors in vitro. In vivo, MLL-ENL leads to expansion of differentiated myeloid cells in our model. High penetrance and long latency of leukemia in our model permits the study of early leukemia development. Our model revealed that MLL-ENL - induced myeloproliferation occurs as early as twelve weeks after MLL-ENL-ERtm activation in the bone marrow and infiltrates the spleen with a consequent decrease in lymphoid B220+CD19+IgM+ cells. Using the TUNEL assay on bone marrow sections, we observed induction of apoptosis in the highly proliferative bone marrow compartment compared to matched controls. These results suggest activation of a potential tumor suppressor mechanism by MLL-ENL in early stages of leukemia. We are currently investigating potential tumor suppressor pathways that might be involved in MLL-ENL - induced apoptosis in preleukemia.

Bioinspired superhydrophobic poly(L‐lactic acid) surfaces control bone marrow derived cells adhesion and proliferation

The aptitude of a cell to adhere, migrate, and differentiate on a compact substrate or scaffold is important in the field of tissue engineering and biomaterials. It is well known that cell behavior can be controlled and guided through the change in micro- and nano-scale topographic features. In this work, we intend to demonstrate that special topographic features that control wettability may also have an important role in the biological performance of biodegradable substrates. Poly(L-lactic acid) surfaces with superhydrophobic characteristics were produced, based on the so-called Lotus effect, exhibiting dual micro- and nano-scale roughness. The water contact angle could be higher than 150 degrees and a value of that order could be kept even upon immersion in a simulated body fluid solution for more than 20 days. Such water repellent surfaces were found to prevent adhesion and proliferation of bone marrow derived cells previously isolated from the femurs of 6-week-old male Wistar rats, when compared with smoother surfaces prepared by simple solvent casting. Such results demonstrate that these superhydrophobic surfaces may be used to control cell behavior onto biodegradable substrates.

Contribution of circulating vascular progenitors in lesion formation and vascular healing: lessons from animal models

It is a widely accepted view that vascular repair results from migration and proliferation of adjacent cells in animal models. On the contrary, accumulating evidence suggests that bone marrow can give rise to endothelial-like cells and smooth muscle like cells that potentially contribute to vascular healing, remodeling, and lesion formation under physiological and pathological conditions. The aim of this article is to review recent findings obtained from animal models of vascular diseases regarding bone marrow derived progenitor cells. Studies using chimeric animals revealed that bone marrow derived cells exist at the sites of vascular healing and lesion formation after injury. High-resolution histological analyses revealed that those bone marrow derived cells do express some markers for endothelial cells or smooth muscle cells. Peripheral mononuclear cells could differentiate into endothelial-like cells or smooth muscle like cells in vitro according to the culture conditions. Circulating progenitors significantly contribute to vascular repair and lesion formation. These findings provide the basis for the development of new therapeutic strategies that involve targeting the mobilization, homing, differentiation, and proliferation of bone marrow- derived vascular progenitor cells.

Treatment of mice with the Ah receptor agonist and human carcinogen dioxin results in altered numbers and function of hematopoietic stem cells

The aryl hydrocarbon receptor (AhR) mediates the carcinogenicity of a family of environmental contaminants, the most potent being 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Increased incidence of lymphoma and leukemia in humans is associated with TCDD exposure. Although AhR activation by TCDD has profound effects on the immune system, precise cellular and molecular mechanisms have yet to be determined. These studies tested the hypothesis that alteration of marrow populations following treatment of mice with TCDD is due to an effect on hematopoietic stem cells (HSCs). Treatment with TCDD resulted in an increased number and proliferation of bone marrow (BM) populations enriched for HSCs. There was a time-dependent decrease in B-lineage cells with a concomitant increase in myeloid populations. The decrease in the B-cell lineage colony-forming unit-preB progenitors along with a transient increase in myeloid progenitors were consistent with a skewing of lineage development from lymphoid to myeloid populations. However, HSCs from TCDD-treated mice exhibited diminished capacity to reconstitute and home to marrow of irradiated recipients. AhR messenger RNA was expressed in progenitor subsets but is downregulated during HSC proliferation. This result was consistent with the lack of response following the exposure of 5-fluorouracil-treated mice to TCDD. The direct exposure of cultured BM cells to TCDD inhibited the growth of immature hematopoietic progenitor cells, but not more mature lineage-restricted progenitors. Overall, these data are consistent with the hypothesis that TCDD, through AhR activation, alters the ability of HSCs to respond appropriately to signals within the marrow microenvironment.

Hypothalamic Proline-Rich Polypeptide Enhances Bone Marrow Colony-Forming Cell Proliferation and Stromal Progenitor Cell Differentiation

The AGAPEPAEPAQPGVY proline-rich peptide (PRP-1) was isolated from neurosecretory granules of the bovine neurohypophysis; it is produced by N. supraopticus and N. paraventricularis. It has been shown that PRP-1 has many potentially beneficial biological effects, including immunoregulatory, hematopoietic, antimicrobial, and antineurodegenerative properties. Here we showed that PRP increased colony-forming cell (CFC) proliferation in rat bone marrow (BM) cells in vivo. In PRP-treated rat BM, the CFU number at day 7 and day 14 was considerably increased in comparison with untreated rat BM and no difference was found at day 21 and day 28. The related peptide [arg8]vasopressin did not reveal CFC proliferation. PRP failed to farther increase CFC proliferation in vitro in BM obtained from PRP-treated or untreated rats. After 3-4 days of human BM stromal cell cultivation in the presence of 2-20 microg/ml PRP the appearance of cells expressing CD15, CD10, CD11a, CD11b, CD3, CD4, and CD16 surface antigens did not differ from the untreated cells. PRP increased the appearance of CD14-positive cells upon 3-4-day incubation with both adult and fetal BM stromal cells. Our results suggest a previously undescribed role for the hypothalamic peptide within neurosecretory hypothalamus-bone marrow humoral axis, because PRP enhances BM colony-forming cell proliferation and stromal cell differentiation.

Erythroid expansion and survival in response to acute anemia stress: The role of EPO receptor, GATA‐1, Bcl‐xL and caspase‐3

Erythropoietic stress occurs under conditions of tissular hypoxia, such as anemia. Functional relationships between erythroid bone marrow (BM) proliferation, differentiation, the expression of survival and apoptotic related proteins, as well as the features of the BM microenvironment upon acute anemic stress, are not fully elucidated. To achieve this aim, CF-1 Swiss mice were injected with a single dose of 5-fluorouracil (5-FU, 150 mg/kg ip) and a multiparametric analysis was conducted for 20 days. Apoptosis (TUNEL assay), BM architecture organization (scanning electronic microscopy), proliferation (DNA assay), differentiation (clonogenic cultures), expression of survival erythroid related proteins (EPO-R, GATA-1, Bcl-xL) as well as the expression of apoptotic- related proteins (Bax, activated Caspase-3) by Western blotting, were evaluated. Experimental data showed that apoptosis, arrest of cell proliferation and disruptions of BM architecture were maximal within the first period of acute stress (1-3 days). Bax and caspase-3 overexpressions were also coincident during this acute period. Moreover, from day 5 upon drug challenge BM responds to acute stress through the EPO-EPO-R system, prompting expressions of GATA-1 and Bcl-xL. Erythroid proliferation rates and red-cell-committed progenitors enhanced in a coordinated way to restore the size and function of the red cell compartment. A second overexpression wave of active caspase-3 was noticed during stress recovery. Together, these results indicate that in response to acute stress a dramatic increase in CFU-E (erythroid colony forming units) population is concomitant with upregulation of EPO-R, GATA-1 and Bcl-xL in the BM erythroid compartment, and that these concurrent processes are crucial for acquiring proper erythroid cell functionality without delayed response to tissular hypoxia.