scholarly journals The Mechanism of the Yigutang-Mediated P13K/AKT/GSK-3β Signal Pathway to Regulate Osteogenic Differentiation of Bone Marrow Stromal Stem Cells to Treat Osteoporosis

2021 ◽  
Vol 2021 ◽  
pp. 1-8
Author(s):  
Ning Li ◽  
Yichen Gong
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Protein Expression ◽  
Bone Tissue ◽  
Normal Group ◽  
Femoral Bone ◽  
Model Group ◽  
Significant Difference ◽  
Gsk 3Β ◽  
The Right

Objectives. To explore the mechanism of Yigutang mediating the P13K/AKT/GSK-3β signaling pathway to regulate the osteogenic differentiation of bone marrow stromal stem cells to treat osteoporosis (OP). Methods. Sixty 12-week-old female SD rats were randomly divided into the normal group, model group, Yigutang group, and estrogen group, with 15 cases in each group. In the model group, Yigutang group, and estrogen group, the ovaries on both sides were removed to construct the model, and the bone mineral density (BMD) of the upper metaphysis of the right femur of the rats in each group was detected. The left femur of each group of rats was removed, and the load-deformation curve of the left femur of each group of rats was calculated. The number of osteoblasts was observed by H&E staining. After extracting the right femurs of rats in each group, real-time fluorescent quantitative PCR and Western blot were used to detect the expression levels of genes and proteins related to the P13K/AKT/GSK-3β signaling pathway. Results. The BMD of the upper metaphysis of the right femur in the Yigutang group and the estrogen group was significantly higher than that of the model group ( P < 0.05 ), while both Yigutang and estrogen groups had no significant difference compared with the normal group ( P > 0.05 ). In addition, there was no significant difference between the Yigutang group and the estrogen group ( P > 0.05 ). The elastic load and maximum load of the Yigutang group and the estrogen group were significantly higher than the model group ( P < 0.05 ), but both were lower than the normal group ( P < 0.05 ). There was no significant difference between the Yigutang group and the estrogen group ( P > 0.05 ). The number of osteoblasts in the Yigutang group and the estrogen group was significantly higher than the model group ( P < 0.05 ), but both were lower than the normal group ( P < 0.05 ). There was no significant difference between the Yigutang group and the estrogen group ( P > 0.05 ). The P13K gene expression in the right femoral bone tissue of rats in the Yigutang group and the estrogen group was significantly higher than that in the model group ( P < 0.05 ), and the AKT gene expression did not change significantly ( P > 0.05 ). The gene expression of GSK-3β was significantly lower than that of the model group ( P < 0.05 ). Compared with the normal group, the gene expression of P13K, AKT, and GSK-3β in the right femur bone tissue of the Yigutang group and the estrogen group did not change significantly ( P > 0.05 ). The protein expression of P13K and P-AKt in the right femoral bone tissue of rats in the Yigutang group and the estrogen group was significantly higher than that of the model group ( P < 0.05 ), and the protein expression of AKT did not change significantly ( P > 0.05 ). The protein expression of GSK-3β was significantly lower than the model group ( P < 0.05 ). Compared with the normal group, the protein expression of P13K, AKT, P-AKt, and GSK-3β in the right femoral bone tissue of the Yigutang group and the estrogen group did not change significantly ( P > 0.05 ). Conclusions. Yigutang can regulate the differentiation of bone marrow stromal stem cells into osteoblasts, which may be achieved by regulating the P13K/AKT/GSK-3β signaling pathway.

scholarly journals Impaired Learning and Memory Ability Induced by a Bilaterally Hippocampal Injection of Streptozotocin in Mice: Involved With the Adaptive Changes of Synaptic Plasticity

2021 ◽  
Vol 13 ◽  
Author(s):  
Cong-Cong Qi ◽  
Xing-Xing Chen ◽  
Xin-Ran Gao ◽  
Jing-Xian Xu ◽  
Sen Liu ◽  
...  
Keyword(s):  
Synaptic Plasticity ◽  
Protein Expression ◽  
Learning And Memory ◽  
Psychiatric Symptoms ◽  
Wnt Signaling Pathway ◽  
Behavioral Performance ◽  
Adaptive Changes ◽  
Memory Ability ◽  
Significant Difference ◽  
Gsk 3Β

Background: Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive cognitive decline, psychiatric symptoms and behavioral disorders, resulting in disability, and loss of self-sufficiency.Objective: To establish an AD-like mice model, investigate the behavioral performance, and explore the potential mechanism.Methods: Streptozotocin (STZ, 3 mg/kg) was microinjected bilaterally into the dorsal hippocampus of C57BL/6 mice, and the behavioral performance was observed. The serum concentrations of insulin and nesfatin-1 were measured by ELISA, and the activation of hippocampal microglia and astrocytes was assessed by immunohistochemistry. The protein expression of several molecular associated with the regulation of synaptic plasticity in the hippocampus and the pre-frontal cortex (PFC) was detected via western blotting.Results: The STZ-microinjected model mice showed a slower bodyweight gain and higher serum concentration of insulin and nesfatin-1. Although there was no significant difference between groups with regard to the ability of balance and motor coordination, the model mice presented a decline of spontaneous movement and exploratory behavior, together with an impairment of learning and memory ability. Increased activated microglia was aggregated in the hippocampal dentate gyrus of model mice, together with an increase abundance of Aβ1−42 and Tau in the hippocampus and PFC. Moreover, the protein expression of NMDAR2A, NMDAR2B, SynGAP, PSD95, BDNF, and p-β-catenin/β-catenin were remarkably decreased in the hippocampus and the PFC of model mice, and the expression of p-GSK-3β (ser9)/GSK-3β were reduced in the hippocampus.Conclusion: A bilateral hippocampal microinjection of STZ could induce not only AD-like behavioral performance in mice, but also adaptive changes of synaptic plasticity against neuroinflammatory and endocrinal injuries. The underlying mechanisms might be associated with the imbalanced expression of the key proteins of Wnt signaling pathway in the hippocampus and the PFC.

Nucleostemin Gene Expression in Acute Promyelocytic Leukemia Patients.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4430-4430
Author(s):  
Farzaneh Ashrafi ◽  
Fatemeh Nadali ◽  
Ardeshir Ghavamzadeh ◽  
Kamran Alimoghaddam ◽  
Shahrbano Rostami ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Acute Promyelocytic Leukemia ◽  
Promyelocytic Leukemia ◽  
Normal Bone Marrow ◽  
Newly Diagnosed ◽  
Normal Bone ◽  
Ns Gene ◽  
Significant Difference

Abstract Abstract 4430 Background Nucleostemin (NS), a novel p53-binding protein has been shown essential for stem and cancer cell proliferation and implicated in oncogenesis. Nucleostemin expression had been shown in gastric cancer (SGC-7901) cells, human hepatocarcinoma (HepG2) cells, human cervical cancer (Hela) cells, human osteosarcoma (OS-732) cells. Aim This work designed to study the NS gene expression in bone marrow cells in acute promyelocytic leukemia (APL) patients and in normal bone marrow specimens. Materials &Methods We examined NS gene expression by Quantitative Real Time PCR in bone marrow specimens of 15 cases of APL patients, before treatment and in 4 bone marrow specimens of healthy donors of bone marrow transplantation. In the same samples of bone marrow aspiration morphology of smears was evaluated. Diagnosis of APL was based on morphology and positive PML/RARA in PCR. RT-PCR used to amplify the NS mRNA, and the GAPDH primer sets used for normalizing. For comparison of NS gene expreesion in 2 groups Mann-Whitney U test was used. Results 15 patients enrolled in this study, 11(73%) newly diagnosed APL and 4(27%) relapsed cases. Mean age of patients was 28.67±9.56 year. NS gene expressed in all bone marrow samples of APL patients. NS gene expressed in normal bone marrow specimens too. NS gene expression in bone marrow of APL patients was significantly higher than normal bone marrows(p value =0.002) Fig 1. There was no significant difference in NS gene expression between newly diagnosed and relapsed APL cases. Discussion According to the results of this study it seems that NS gene expressed in normal marrow. NS expression in adult bone marrow hematopoietic stem cells had been reported in previous reports and it had been shown that NS does not express in granulocytes and B lymphocytes. It seems that stem cells and proliferating cells in the normal marrow are the source of NS expression detected in normal marrow. NS expreesion in bone marrow of APL patients was significantly higher than normal marrow. In these patients before treatment marrow is replaced by undifferentiated blasts and promyelocytes. We concluded that NS expression in these cells were high. It had been shown that NS down regulation may lead to cell cycle exit. High expression of NS in APL patients can be used in future researches for finding new targeted therapies in this disease. Disclosures: No relevant conflicts of interest to declare.

Role for PP2A Regulatory Subunit B55α as A Regulator of AKT and Other Signaling Pathways In AML.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1668-1668
Author(s):  
Peter P. Ruvolo ◽  
YiHua Qui ◽  
Kevin R Coombes ◽  
Nianxiang Zhang ◽  
Vivian Ruvolo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Protein Expression ◽  
Normal Bone Marrow ◽  
Protein Levels ◽  
B Subunit ◽  
Normal Bone ◽  
Cd34 Cells ◽  
Blast Cells ◽  
B Subunits

Abstract Abstract 1668 Activation of survival kinases such as Protein Kinase B (AKT), Protein Kinase C (PKC), and Extracellular Receptor Activated Kinase (ERK) predict poor clinical outcome for patients with acute myeloid leukemia (AML; Kornblau et al Blood 2006). A better understanding of how the activities of these kinases are regulated by phosphorylation and dephosphorylation will enable the development of targeted therapies directed against this axis. Protein Phosphatase 2A (PP2A) negatively regulates PKC, AKT, and ERK but its role in AML is not clear. In the current study we examined the role of PP2A in regulating AKT in AML. Activation of AKT involves phosphorylation of threonine 308 (T308) and serine 473 (S473). A recent study has indicated that phosphorylation of AKT at T308 but not S473 is a poor prognostic factor for AML patients and that PP2A activity negatively correlated with T308 phosphorylation (Gallay et al Leukemia 2009). PP2A is a family of different isoforms that form hetero-trimers consisting of a catalytic C subunit, a scaffold A subunit, and one of at least 21 different regulatory B subunits. The functionality of each PP2A isoform is determined by the regulatory B subunit. Thus to understand PP2A regulation of AKT in AML, it is essential to study the B subunit that regulates the AKT phosphatase. The PP2A isoform regulating AKT in the AML patients is currently unknown. Evidence suggests that the B55a subunit is responsible for dephosphorylation of AKT at T308. In the current study, we compared B55α gene expression in blast cells derived from AML patients with normal counterpart (i.e. CD34+) cells derived from normal bone marrow donors by real time PCR. Surprisingly, B55α gene expression was higher in the patients. Reverse Phase Protein Analysis (RPPA) is a powerful tool that allows for the analysis of protein expression from patient samples. Protein levels of the PP2A B subunit were analyzed by RPPA in AML blast cells obtained from 511 newly diagnosed AML patients and CD34+ cells obtained from 11 normal bone marrow donors. Levels of B55α protein were significantly lower in the blast cells from the AML patients compared to normal CD34+ cells. While the mechanism for the observed difference in gene versus protein expression in the leukemia cells has yet to be determined, a plausible mechanism is that the B55α protein is being proteolyzed since monomeric PP2A B subunits that are not part of the PP2A hetero-trimer are degraded. Importantly the reduced levels of B55α protein observed would be predicted if AKT were activated in the AML blast cells. We next compared AKT phosphorylation status with B55α protein expression in the AML blast cells using RPPA to answer this question. Analysis of RPPA data revealed that there was no correlation between B55α protein levels and levels of total AKT protein or with levels of AKT phosphorylated at S473 in the AML samples. However, there was a moderate but significant negative correlation between B55α protein levels and levels of AKT phosphorylated at T308. This result suggests that B55α is mediating dephosphorylation of AKT at T308 but not S473 in the AML cells. B55α expression was not associated with FAB classification but was positively correlated with high blast and peripheral blood counts. While the level of expression of the B subunit did not correlate with overall survival, intermediate levels of B55α expression were associated with longer complete remission duration. We predict that higher levels of B55α would reflect low levels of other PP2A B subunits. Consistent with this prediction, B55α expression positively correlated with MYC expression in the AML patients. MYC expression is regulated by a B subunit that competes with B55α (i.e. B56α). These findings suggest that B55α may play an important role in AML as a negative regulator of AKT and perhaps by other as yet unidentified functions. Activation of B55α is a potential therapeutic target for overcoming the AKT activation frequently observed in AML. Disclosures: No relevant conflicts of interest to declare.

Mesenchymal Stromal Cells From Myelodysplastic Syndrome Patients Show Lower DICER1 and DROSHA Expression, as Well as Decreased Mir-155 and Mir-181a MicroRNA Expression, When Compared with Healthy Controls

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 397-397
Author(s):  
Carlos Santamaría ◽  
Olga López-VIllar ◽  
Sandra Muntión ◽  
Belén Blanco ◽  
Soraya Carrancio ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Protein Expression ◽  
Myelodysplastic Syndrome ◽  
Western Blot ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Microrna Expression ◽  
Healthy Controls ◽  
Lower Expression

Abstract Abstract 397 Mesenchymal stromal cells (MSC) are closely related to the regulation of hematopoietic stem cell niche. Recently, Raaijmakers et al (Nature, 2010), published that deletion of Dicer1, a RNase III enzyme involved in microRNA biogenesis, in murine MSC-derived osteoprogenitors triggered peripherical blood cytopenias, myelodysplasia and subsequent AML, showing that molecular alterations in bone marrow microenvironment could result in clonal impaired haematopoiesis. Here, we have investigated whether MSC from myelodysplastic syndrome (MDS) patients show differences in DICER1 and DROSHA, another RNA III endonuclease, in comparison to healthy MSC. In addition, we have analyzed several hematopoietic-related microRNAs in these same samples. Bone marrow MSC from MDS patients (n=35; 10 5q- syndrome, 4 RA, 5 RARS, 10 RCMD, 3 RAEB, 2 MDS-U and 1 hypocellular MDS) and healthy donors (HD, n=20) were isolated and in vitro expanded following standard procedures until the third passage. Additionally, paired mononuclear cells (MNC) from 13 MDS and 8 HD were obtained. Total RNA was isolated using TRIzol reagent (Invitrogen). DICER1 and DROSHA relative gene expressions were assessed by quantitative PCR (Q-PCR) using commercial TaqMan® assay (Applied Biosystems®) with GAPDH as control gene. DICER1 and DROSHA (Abcam) protein expression were evaluated in whole cell lysates by western blot, using calnexin (Stressgen) as control. Several microRNAs with known role in hematopoiesis and immune system regulation were analyzed in 25 MDS and 12 HD by Q-PCR using commercial TaqMan® MicroRNA assay (Applied Biosystems®) with RNU43 as control microRNA. MSC from MDS showed significant lower DICER1 (0.0035±0.0020 vs. 0.0076±0.0092; p=0.044) and DROSHA (0.0070±0.0028 vs. 0.0135±0.0176; p=0.019) gene expression levels than healthy controls. Moreover, MSC from MDS showed lower protein expression of both DICER1 and DROSHA by western blot analysis, confirming Q-PCR findings. By contrast, no difference in either DICER1 (0.0197±0.0151 vs. 0.0173±0.0112; p=0.9) or DROSHA (0.0089±0.0023 vs. 0.0067±0.0037; p=0.09) gene expression were observed between MNC from MDS and HD. As far as microRNA expression, we observed a lower expression of mir-155 (0.63±0.92 vs. 0.94±0.49; p=0.007) and mir-181a (1.30±0.95 vs. 2.02±1.05; p=0.041) in MSC from MDS in comparison to healthy controls. Mir-155 and mir-181a are involved in T-cell and B-cell differentiation, while mir-155 are also related to erythroid and megakarycytic differentiation. We conclude that MSC from MDS patients show lower expression of DICER and DROSHA, two relevant RNA-III endonucleases involved in the microRNA biogenesis, confirming recent findings in murine models. Moreover, the expression of some microRNA is impaired in these cells, raising the possibility that these microenvironmental alterations could be involved in the MDS pathophysiology. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Regulation of MHC class II gene expression in macrophages by hematopoietic colony-stimulating factors (CSF). Induction by granulocyte/macrophage CSF and inhibition by CSF-1.

1989 ◽  
Vol 170 (5) ◽  
pp. 1559-1567 ◽  
Author(s):  
C L Willman ◽  
C C Stewart ◽  
V Miller ◽  
T L Yi ◽  
T B Tomasi
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Protein Expression ◽  
Mhc Class Ii ◽  
Cellular Proliferation ◽  
Ifn Gamma ◽  
Gm Csf ◽  
Ifn Alpha ◽  
Gene And Protein Expression ◽  
Alpha Beta

CSF-1 and granulocyte/monocyte CSF (GM-CSF) were shown to modulate the levels of Ia gene and protein expression in bone marrow-derived macrophages (BMM). Recombinant GM-CSF induced high levels of Ia expression, similar to the levels induced by INF-gamma, while IL-3 had no effect. In contrast, recombinant CSF-1 not only suppressed the basal levels of Ia gene and protein expression in BMM, but also inhibited the induction of Ia by IFN-gamma and GM-CSF. Basal levels of Ia were not inhibited by recombinant CSF-1 until after 16-24 h of culture, suggesting an indirect mechanism of suppression. IFN-alpha/beta and PGE2 were shown not to be involved in the CSF-1 inhibition of basal levels of Ia expression. However, the CSF-1-mediated suppression of both the basal levels of Ia expression and the induction of Ia in BMM by IFN-gamma and GM-CSF did correlate with the induction of cellular proliferation. These data imply that in addition to regulating hematopoiesis, CSFs may regulate the initiation of the immune response through their effects on Ia expression in macrophages.

Research on the Mechanism of Asiaticoside Inhibiting Osteoclast Differentiation and Promoting the Recovery of Steroid-Induced Osteonecrosis of the Femoral Head (SIONFH)

2021 ◽  
Vol 11 (8) ◽  
pp. 1606-1611
Author(s):  
Meijing Miao ◽  
Liping Guo ◽  
Pengfei Su ◽  
Jinshan Ji ◽  
Baoli Li
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Femoral Head ◽  
Protein Expression ◽  
Western Blot ◽  
Osteoclast Differentiation ◽  
Culture Fluid ◽  
Qrt Pcr

Our study aims to assess whether asiaticoside promotes the recovery of SINOFH by inhibiting bone marrow stem cells (BMSCs) differentiation into osteoclasts (OC). BMMs were induced to form OC system by dexamethasone in vitro and ELISA detected the expression of OC-related genes formation by asiaticoside. BMSCs were cultured followed by analysis of BMSCs morphology under microscope, gene expression by qRT-PCR. TRACP and c-Src level by western blot, RANKL, OPG and TRACP5b level by ELISA. Asiaticoside inhibited the expression of OC formation in SIONFH. The expression of OC-related genes increased with the induction days. With the increasing of induction days, asiaticoside level in culture fluid was decreased. While after asiaticoside interference, OCrelated genes and proteins levels were significantly down-regulated. Aasiaticoside can significantly increase the RANKL signaling protein expression. In conclusion, asiaticoside promotes the recovery of SINOFH by inhibiting BMSCs differentiation into OC.

Influence of Maternal Folic Acid Supplementation on Hematopoietic Factors in Offspring.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4276-4276
Author(s):  
Kimberly J. Johnson ◽  
David A. Largaespada ◽  
Tucker W. LeBien ◽  
Raha Allaei ◽  
Julie A. Ross
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Folic Acid ◽  
Lymphoblastic Leukemia ◽  
Surface Marker ◽  
Control Group ◽  
Offspring Survival ◽  
Childhood All ◽  
Significant Difference ◽  
B Lineage

Abstract Observational studies of childhood acute lymphoblastic leukemia (ALL) have generally supported a protective role for maternal intake of foods or vitamins rich in folic acid during pregnancy. Using C57BL/6J mice, we evaluated the effect of dietary folic acid intake in the perigestational period on surface marker and gene expression in B-lineage cells as a potential model to further elucidate the role of maternal diet in the etiology of childhood ALL. Thirty-six female mice were randomized to one of three folic-acid varied diets (low=0.3, control=2.0, high=8.0 mg/kg) one month prior to mating and maintained on the diet until pup weaning at three weeks. Reproductive characteristics and offspring red blood cell (RBC) folate were recorded, and bone marrow lymphocytes were isolated for surface marker characterization by flow cytometry. For gene expression analyses, CD19 positive B-lineage bone marrow cells were purified from three pups per dietary group using anti-CD19 microbeads. Gene expression levels were interrogated using the Agilent mouse whole genome microarray. No statistically significant differences were found among dietary groups with respect to mating success, litter size, estimated 21 day offspring weights, or the percentage of male offspring. However, there were significant differences among the 3 diets in offspring survival to the end of the experiment (39%, 84%, and 30% for the 0.3, 2.0, and 8.0 mg/kg groups, respectively). Pup RBC folate values were positively correlated with dietary folic acid dose with mean values of 428, 615, and 724 ng/ml, respectively. Mice were euthanized at 3 weeks. There was no significant difference in the expression of CD19, IgM, or kappa light chains on bone marrow lymphocytes in the offspring among dietary groups. ANOVA analysis, using Genedata Expressionist Pro software, showed differential expression of 267 genes using a Benjamini and Hochburg false discovery rate cutoff of 10%. Three main patterns of gene expression between pups of different dietary groups were identified through unsupervised hierarchical clustering analysis; 152 and 86 genes were up- and down-regulated, respectively, in both the high and the low folic acid groups relative to the control group. Twenty-seven genes showed increasing expression with increasing dietary folic acid dose. In conclusion, dietary folic acid given to dams prior to conception through weaning had a significant effect on offspring survival and gene expression. Additional analyses are underway to determine the relevance of genes found to be differentially expressed with respect to risk of B-cell lineage leukemias followed by validation of relevant gene candidates through quantitative RT-PCR. These data will be used to help inform future etiologic studies of childhood ALL.

Oxygen Tension in the Bone Marrow (BM) of Patients with Malignant and Indolent Monoclonal Gammopathy: Role of Hypoxia and Hypoxia-Inducible Factor (HIF)-1α in the Regulation of Gene Expression and Pro-Angiogenic Profiles of CD138+ Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 422-422 ◽  
Author(s):  
Nicola Giuliani ◽  
Simona Colla ◽  
Paola Storti ◽  
Valentina Sgobba ◽  
Katia Todoerti ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Bone Marrow ◽  
Monoclonal Gammopathy ◽  
Hypoxia Inducible Factor ◽  
Heme Oxygenase 1 ◽  
Protein Ubiquitination ◽  
Significant Difference ◽  
Bone Biopsies

Abstract Abstract 422 Multiple myeloma (MM) is characterized by the accumulation of malignant plasmacells into the bone marrow (BM) microenvironment that supports their growth and survival. Particularly, an increase of BM angiogenesis occurs in relationship with plasmacells infiltration playing a critical role in the progression on monoclonal gammopathy. Hypoxia is known to be associated to angiogenesis in solid tumors as well as the hypoxia-inducible factor (HIF)-1α is a critical trigger and regulator of the angiogenic switch. Actually, the oxygen levels in the BM of patients with monoclonal gammopathy as well the effects of hypoxia and HIF-1α on the gene expression and pro-angiogenic profile of CD138+ cells are not known. In this study, first, we investigated the level of BM oxygen saturation (sO2) and partial pressure (pO2) in a cohort of 44 patients with monoclonal gammopathy at the diagnosis including active MM (n°=25), smoldering MM (n° = 8) and MGUS (n°=11) showing that BM of MM patients was hypoxic (mean ± SD of pO2: 52.5 ± 8.69 mmHg; sO2 of 83.7 ± 11%) even if any significant difference in both pO2 and sO2 was not observed in comparison with smoldering MM patients or MGUS as well as in relationship with the stage of disease (p=0.7). Next, we evaluated how hypoxia exposition could modify the gene expression profile of CD138+cells isolated form MM patients by U133 Plus2.0 Arrays. By supervised analysis performed on 11 paired samples we found that hypoxia significantly modulated 714 genes in CD138+ cells isolated from MM patients. Interestingly, genes belonging either to oxidative stress and hypoxia signaling, including heme oxygenase 1 (HMOX1) and the heat shock proteins 90kDa alpha (HSP90AA1; HSP90AB1), or to protein ubiquitination pathway, such as XIAP, were significantly up-regulated by hypoxia. Among the pro-angiogenic genes, we found that both VEGFA and IL8 were induced by hypoxia. Following, given that HIF-1α accumulates only in hypoxic condition, we checked HIF-1α protein expression in the BM of MM patients by immunohistochemistry on bone biopsies immediately after fixation. A strong HIF-1α immunostaining was demonstrated in MM cells at nuclear level in all patients analyzed. Interestingly, the presence of HIF-1α protein was also observed in isolated CD138+ MM cells of about 28% of MM patients in normoxic condition. In order to investigate the potential role of HIF-1α on MM cell gene expression and angiogenic profile we performed HIF-1α silencing in MM cells by siRNA anti-HIF-1α and that exposed cells to normoxic or hypoxic conditions. HIF-1α suppression was associated to the modulation of pro-angiogenic molecules (VEGFA, VEGFB, IL-8 and PGF) and oxidative stress (SOD2, PTGS2, TXT) and glycogenolysis regulating (ENO2, ALDOC, PFKFB3, HK2, PDK1, PFKFB4) genes. Data obtained were than validated by real time PCR and at protein level by western blot and ELISA assay. Consistently we found that HIF-1α suppression significantly inhibited the pro-angiogenic properties, evaluated in an in vitro angiogenesis model as capillary junctions and tubules formation and tubule length. In conclusion, we demonstrate that the MM-BM environment is hypoxic and that HIF-1α protein expressed by MM cells. Consistently we show that hypoxia and HIF-1α significantly modulate the gene expression profiles of MM cells regulating the expression of the pro-angiogenic factors by MM cells and their pro-angiogenic properties. Disclosures: No relevant conflicts of interest to declare.

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