Role of endothelial nitric oxide in bone marrow-derived progenitor cell mobilization

Mobilization and recruitment of bone marrow-derived progenitor cells (BMDPCs) play an important role in postischemic tissue repair. Patients with coronary artery disease (CAD) or peripheral vascular disease (PVD) exhibit endothelial dysfunction, and as a result are likely to have a reduced number of progenitor cells mobilized in their peripheral circulation following ischemic injury. Identification of eNOS independent pathways for BMDPC mobilization may have important therapeutic value in this patient population. To identify such mechanisms we investigated the effect of granulocyte-colony stimulating factor (GCSF) and stem cell factor (SCF) in eNOS-KO mice with and without surgical hind-limb ischemia. Our results suggest that BMDPC mobilization can be achieved via activation of NO-independent pathways.     

黄芪提取物对大鼠骨髓源性内皮祖细胞的作用

摘要： 目的 探讨黄芪提取物对大鼠骨髓源性内皮祖细胞 （EPCs） 黏附、 迁移、 活力、 血管形成能力及内皮型一氧 化氮合成酶 （eNOS） 表达的影响。方法 体外培养、 分离和鉴定 EPCs, 设 10-4、 10-3、 10-2 g/L 黄芪提取物组和对照组。 倒置显微镜下观察并比较各组 EPCs 黏附、 迁移、 血管形成能力的差异; MTT 法检测 EPCs 的活力变化; RT-PCR 法检 测 EPCs 中 eNOS mRNA 的表达;Western blot 检测 EPCs 中 eNOS 蛋白的表达。结果 与对照组相比, 不同浓度黄芪 提取物组促 EPCs 黏附、 迁移、 血管形成能力均显著增强, 且呈浓度依赖性(F 值分别为 15.256、 13.633、 97.549, 均 P < 0.05); EPCs 的活力显著增加, 呈时间 （F 时间=9.755） 和浓度依赖性 （F 组间=10.018）; 且 EPCs 中 eNOS mRNA 和蛋白的表 达水平均明显升高, 呈浓度依赖性(F 值分别为 56.356、 77.125, 均 P < 0.05)。结论 黄芪提取物具有调控 EPCs 促血 管新生的作用, 该作用可能与上调eNOS 的表达水平密切相关。     

蛋白激酶D1在大鼠骨髓源性内皮祖细胞中的促血管新生作用

目的探讨蛋白激酶D1(PKD1)对大鼠骨髓源性内皮祖细胞(EPCs)黏附、迁移、增殖、血管形成能力及内皮型一氧化氮合成酶(e NOS)表达的影响。方法体外培养、分离和鉴定大鼠骨髓源性EPCs,观察PKD1及其特异性阻断剂CID755673对EPCs的黏附、迁移、增殖、血管形成能力的影响,以及对EPCs中e NOS的mRNA表达和蛋白表达的影响。结果 EPCs体外细胞培养实验表明,PKD1可明显促进EPCs的黏附、迁移和增殖,提升EPCs的血管形成能力,上调EPCs中e NOS的mRNA表达和蛋白表达水平。结论 PKD1具有调控EPCs促血管新生的作用,其促血管新生的作用可能以一种依赖e NOS的方式进行。     

Concerns and hopes for stem cell therapy in cardiology: focus on endothelial progenitor cells

The crucial role played by the endothelium in cardiovascular disorders has been repetitively recognised. Endothelium injury has been implicated in atherosclerosis, thrombosis, hypertension and other cardiovascular diseases. Recently, however, research has undertaken a new avenue. As mature endothelial cells posses limited regenerative capacities, the interest has been switched to the circulating endothelial progenitor cells (EPCs). Indeed, the scientific community has made progress in understanding the role of EPCs in the maintenance of endothelial integrity and function as well as post natal neovascularisation. It has been suggested that these cells are able to home in the site of heart injury / damage and that they might take part in angiogenesis, giving hope for new treatment opportunities. There is evidence that reduced availability of EPCs or impairment of their function is associated with more severe CV disease and to comorbid risk factors. Different current drug regimes are able to influence bone marrow production and release of EPCs and several growth factors are considered for possible useful new therapeutic approaches. Thus, many studies into the potential use of EPCs in the clinical setting have recently been conducted with conflicting results. The goal of this review article is to discuss current therapies to regenerate new vessels and therefore to enhance myocardial function. The article overviews the search strategy and the pathophysiological aspects behind this therapy, consider the target currently under investigation and set the stage for new ideas.     

Decreased mobilization of endothelial progenitor cells contributes to impaired neovascularization in diabetes

• 1 Circulating bone marrow (BM)‐derived endothelial progenitor cells (EPCs) play an important role in neovascularization. In the present study, we investigated the mechanisms underlying the reduction in circulating EPCs in a mouse model of diabetes induced by streptozotocin.
• 2 Compared with non‐diabetic controls, diabetic mice had reduced circulating EPCs (0.59 ± 0.11 vs 0.94 ± 0.21%, respectively; P < 0.01) and increased plasma endothelial microparticles (18 642 ± 6809 vs 5692 ± 1862/mL, respectively; P < 0.01). In a mouse bone marrow (BM) transplantation model, increased adhesion of transplanted BM cells to aortas of diabetic mice was observed compared with control (900 ± 194 vs 431 ± 109 cells/mm², respectively; P < 0.01).
• 3 Following hindlimb ischaemia, diabetic mice exhibited suppressed EPC mobilization, a reduction in the expected increase in capillary density and suppressed restoration of transcutaneous oxygen pressure in the ischaemic tissue. Diabetic mice also showed impaired ischaemia‐induced upregulation of vascular endothelial growth factor (VEGF), hypoxia‐inducible factor (HIF)‐1α and interleukin‐1β, an exaggerated increase in matrix metalloproteinase (MMP)‐2 and ‐9 and a suppressed increase in tissue inhibitor of matrix metalloproteinase (TIMP)‐1. On multivariate analysis, VEGF expression was the only independent factor related to circulating EPC count.
• 4 In conclusion, the data indicate that the decrease in basal circulating EPCs in diabetes may be attributable, in part, to consumptive loss of EPCs due to increased endothelial damage. Impairment of ischaemia‐induced EPC mobilization in the diabetic mouse model is associated with altered HIF‐1α/VEGF and MMP/TIMP regulation and represents a novel mechanism underlying defective postischaemic neovascularization in diabetes.

     

Autotransplantation of bone marrow-derived stem cells as a therapy for neurodegenerative diseases

Neurodegenerative diseases are characterized by a progressive degeneration of selective neural populations. This selective hallmark pathology and the lack of effective treatment modalities make these diseases appropriate candidates for cell therapy. Bone marrow-derived mesenchymal stem cells (MSCs) are self-renewing precursors that reside in the bone marrow and may further be exploited for autologous transplantation. Autologous transplantation of MSCs entirely circumvents the problem of immune rejection, does not cause the formation of teratomas, and raises very few ethical or political concerns. More than a few studies showed that transplantation of MSCs resulted in clinical improvement. However, the exact mechanisms responsible for the beneficial outcome have yet to be defined. Possible rationalizations include cell replacement, trophic factors delivery, and immunomodulation. Cell replacement theory is based on the idea that replacement of degenerated neural cells with alternative functioning cells induces long-lasting clinical improvement. It is reasoned that the transplanted cells survive, integrate into the endogenous neural network, and lead to functional improvement. Trophic factor delivery presents a more practical short-term approach. According to this approach, MSC effectiveness may be credited to the production of neurotrophic factors that support neuronal cell survival, induce endogenous cell proliferation, and promote nerve fiber regeneration at sites of injury. The third potential mechanism of action is supported by the recent reports claiming that neuroinflammatory mechanisms play an important role in the pathogenesis of neurodegenerative disorders. Thus, inhibiting chronic inflammatory stress might explain the beneficial effects induced by MSC transplantation. Here, we assemble evidence that supports each theory and review the latest studies that have placed MSC transplantation into the spotlight of biomedical research.     

Lineage-related and particle size-dependent cytotoxicity of chitosan nanoparticles on mouse bone marrow-derived hematopoietic stem and progenitor cells

Chitosan nanoparticles (CSNPs) have potential applications in stem cell research. In this study, ex vivo cytotoxicity of CSNPs on mouse bone marrow-derived (MBMCs) hematopoietic stem and progenitor cells (HSPCs) was determined. MBMCs were exposed to CSNPs of different particle sizes at various concentrations for up to 72 h. Cytotoxicity effect of CSNPs on MBMCs was determined using MTT, Live/Dead Viability/Cytotoxicity assays and flow cytometry analysis of surface antigens on HSCs (Sca-1⁺), myeloid-committed progenitors (CD11b⁺, Gr-1⁺), and lymphoid-committed progenitors (CD45⁺, CD3e⁺). At 24 h incubation, MBMCs' viability was not affected by CSNPs. At 48 and 72 h, significant reduction was detected at higher CSNPs concentrations. Small CSNPs (200 nm) significantly reduced MBMCs' viability while medium-sized particle (∼400 nm) selectively promoted MBMCs growth. Surface antigen assessment demonstrated lineage-dependent effect. Significant decrease in Sca-1⁺ cells percentage was observed for medium-sized particle at the lowest CSNPs concentration. Meanwhile, reduction of CD11b⁺ and Gr-1⁺ cells percentage was detected at high and intermediate concentrations of medium-sized and large CSNPs. Percentage of CD45⁺ and CD3e⁺ cells along with ROS levels were not significantly affected by CSNPs. In conclusion, medium-sized and large CSNPs were relatively non-toxic at lower concentrations. However, further investigations are necessary for therapeutic applications.     

Sonic hedgehog protein promotes bone marrow-derived endothelial progenitor cell proliferation, migration and VEGF production via PI 3-kinase/Akt signaling pathways

AIM: To investigate the effects of Sonic hedgehog (shh) protein on bone marrow- derived endothelial progenitor cells (BM-EPC) proliferation, migration and vascular endothelial growth factor (VEGF) production, and the potential signaling pathways involved in these effects. METHODS: Bone marrow-derived Flk-1(+) cells were enriched using the MACS system from adult Kunming mice and then BM-EPC was cultured in gelatin-coated culture dishes. The effects of shh N-terminal peptide on BM-EPC proliferation were evaluated using the MTT colorimetric assay. Cell migration was assayed using a modified Boyden chamber technique. The production of VEGF was determined by ELISA and immunofluorescence analysis. The potential involvement of PKC and PI3K signaling pathways was explored using selective inhibitor or Western blot. RESULTS: The proliferation, migration and VEGF production in BM-EPC could be promoted by endogenous shh N-terminal peptide at concentrations of 0.1 microg/mL to 10 microg/mL, and could be inhibited by anti-shh antibodies. Shh-mediated proliferation and migration in BM-EPC could be partly attenuated by anti-VEGF. Phospho-PI3-kinase expression in newly separated BM-EPC was low, and it increased significantly when exogenous shh N-terminal peptide was added, but could be attenuated by anti-human/mouse shh N-terminal peptide antibody. Moreover, the inhibitor of the PI3-kinase, but not the inhibitor of the PKC, significantly inhibited the shh-mediated proliferation, migration and VEGF production. CONCLUSION: Shh protein can stimulate bone marrow-derived BM-EPC proliferation, migration and VEGF production, which may promote neovascularization to ischemic tissues. This results also suggests that the PI3-kinase/Akt signaling pathways are involved in the angiogenic effects of shh.     

阿托伐他汀对兔循环内皮祖细胞数和心肌血管新生的影响

目的：观察兔心肌梗死模型中不同剂量阿托伐他汀对循环内皮祖细胞数量和心肌血管新生的影响。方法：建立新西兰大耳白兔心肌梗死模型后,动物随机分成生理盐水[5 mg/（kg.d）]对照组、阿托伐他汀常规剂量组[5 mg/（kg.d）]和阿托伐他汀强化剂量组[20 mg/（kg.d）],灌胃给药8周。取外周血分离单核细胞进行内皮祖细胞培养1周后,鉴定FITC-UEA-Ⅰ和Di1-acLDL双染色阳性细胞为正在分化的内皮祖细胞,在倒置荧光显微镜下计数。以羊抗兔CD31抗体对心肌微血管进行免疫组化染色,显微镜下计数心肌新生的微血管数,取血管数平均值为微血管密度。结果：与对照组和强化剂量组比较,阿托伐他汀常规剂量组明显增加外周血中内皮祖细胞的数量,高倍视野中对照组、强化剂量组和常规剂量组内皮祖细胞数分别为（211.17±18.65）、（240.29±44.37）和（321.44±30.27）个（P〈0.05）,强化剂量组与对照组比较差异无统计学意义。常规剂量组心肌微血管密度较对照组和强化剂量组明显升高,高倍视野中3组微血管数分别为（16.78±3.50）、（11.17±2.64）和（12.86±3.72）（P〈0.05）。强化剂量组与对照组比较虽有升高趋势,但差异无统计学意义。结论：常规剂量阿托伐他汀可增加心肌梗死后兔外周循环血中内皮祖细胞数量,促进缺血坏死心肌内新生血管的形成,这些作用可能独立于其降脂效应。     

人脐带间充质干细胞对小鼠溃疡性结肠炎的改善作用

目的 探讨人脐带来源的间充质干细胞(HUC-MSCs)对小鼠溃疡性结肠炎(UC)的作用.方法 小鼠自由饮用3.5％ 葡聚糖硫酸钠水溶液构建UC模型.按照体重将小鼠随机分为6组:正常组、模型组、对照组和低、中、高3个剂量实验组,每组10只.正常组与模型组腹腔注射高糖DMEM培养液;对照组给予美沙拉嗪100 mg·kg-1...     

骨髓间充质干细胞对胶质瘤C6细胞增殖的影响

目的探讨大鼠骨髓间充质干细胞(MSCs)对胶质瘤C6细胞体外增殖的影响。方法从大鼠骨髓中分离MSCs,体外培养和鉴定。取MSCs培养上清液,按照1/5,2/5,3/5的比例加入到C6培养基中,用MTT实验和BrdU标记指数(BrdU LI)评估C6细胞增殖。将MSCs与C6按照1∶1,1∶2,2∶1比例共培养。结果大鼠MSCs贴壁生长,有较强的增殖能力。经硫代甘油和巯基乙醇诱导分化后,分化为神经元或胶质细胞样细胞。C6培养基中加入MSCs培养上清液后,活性受到明显抑制,BrdU LI明显减低(P<0.05)。C6与MSCs共培养后,生长明显受到抑制,BrdU LI明显降低,尤其在MSCs周围,表现更加明显。结论大鼠MSCs在体外有明显的抑制胶质瘤C6增殖作用。     

Behaviour and ultrastructure of human bone marrow-derived mesenchymal stem cells immobilised in alginate-poly-l-lysine-alginate microcapsules

Abstract

Context: Human bone marrow mesenchymal stem cells (hBM-MSCs) show a great promise for the treatment of a variety of diseases. Despite the previous trials to encapsulate hBM-MSCs in alginate-poly-l-lysine-alginate (APA) systems, the various changes that follow immobilisation have not been ascertained yet. Objective: Determine the various consequences derived from entrapment on cell behaviour, putting special emphasis on the ultrastructure. Methods: hBM-MSCs were immobilised in APA microcapsules to further characterise their viability, metabolic activity, proliferation, VEGF-secretability, and morphology. Results: The VEGF produced by monolayer hBM-MSCs increased significantly 1 d post-encapsulation, and was maintained for at least 4 weeks. TEM imaging of cells revealed well preserved ultrastructure indicating protein synthesis and high metabolic activity. Conclusion: Although APA microencapsulation did not support 100% of fully viable hBM-MSCs for long-term cultures, it was conceived to enhance both VEGF secretion and metabolic activity while not losing their stemness characteristics.     

Voltage-gated K+ channels in adipogenic differentiation of bone marrow-derived human mesenchymal stem cells

Aim:

To determine the presence of voltage-gated K⁺ (Kv) channels in bone marrow-derived human mesenchymal stem cells (hMSCs) and their impact on differentiation of hMSCs into adipocytes.

Methods:

For adipogenic differentiation, hMSCs were cultured in adipogenic medium for 22 d. The degrees of adipogenic differentiation were examined using Western blot, Oil Red O staining and Alamar assay. The expression levels of Kv channel subunits Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv2.1, Kv3.1, Kv3.3, Kv4.2, Kv4.3, and Kv9.3 in the cells were detected using RT-PCR and Western blot analysis.

Results:

The expression levels of Kv2.1 and Kv3.3 subunits were markedly increased on d 16 and 22. In contrast, the expression levels of other Kv channel subunits, including Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv4.2, Kv4.3, and Kv9.3, were decreased as undifferentiated hMSCs differentiated into adipocytes. Addition of the Kv channel blocker tetraethylammonium (TEA, 10 mmol/L) into the adipogenic medium for 6 or 12 d caused a significant decrease, although not complete, in lipid droplet formation and adipocyte fatty acid-binding protein 2 (aP₂) expressions. Addition of the selective Kv2.1 channel blocker guangxitoxin (GxTX-1, 40 nmol/L) into the adipogenic medium for 21 d also suppressed adipogenic differentiation of the cells.

Conclusion:

The results demonstrate that subsets of Kv channels including Kv2.1 and Kv3.3 may play an important role in the differentiation of hMSCs into adipocytes.     

内皮祖细胞骨移植在缺血再灌注肾损伤中的促血管新生作用

缺血/再灌注(I/R)肾损伤是急性肾衰竭(ARF)的最常见病因.研究证实,内皮祖细胞(EPC)在血管新生和内皮再生,尤其在缺血性疾病治疗方面具有重要意义.最近对EPC在I/R损伤时所发挥的作用进行了探讨,取得了一定进展.本文综述I/R肾损伤后EPC的促血管新生作用.     

Mast cells and basophils: trojan horses of conventional lin- stem/progenitor cell isolates

Cancer microenvironment is increasingly recognized as an important factor affecting cancer onset and progression. Since Wirchow reported in 1863 that tumors contain inflammatory cells, the field shifted significantly forward, and immune cells residing in tumors appear to be attractive targets of cancer therapies. For some methods, such as stem/progenitor cell isolation from both cancer and healthy tissues, removal of contaminating immune cells is crucial to achieve consistent, reproducible and accurate results. Despite current methods of lineage negative selection accounts for removal of over 99 % of immune cells from stem/progenitor cell isolates, the vast majority of lineage antibody cocktails retain basophils, dendritic cells, and mast cells. Here we discuss the ability of the most commonly used lineage markers to bind to the plasma membrane of mast cells and/or basophils, and suggest alternatives, which may be used for negative selection of these cellular populations. Both, mast cells and basophils, were shown to participate actively in cancer-associated angiogenesis, tissue remodeling and recruitment of other immune cell types, including eosinophils, B cells, memory T cells and Treg cells. In turn, tumor-derived peptides and chemotactic factors are known to recruit and activate mast cells in neoplasias, resulting in altered tumor progression. Repeated findings of CD34+ populations of mast cells and basophils further highlight necessity of their separation from stem/progenitor cell isolates in both, preclinical experiments and clinical praxis.     

Zinc oxide nanoparticles induce migration and adhesion of monocytes to endothelial cells and accelerate foam cell formation

Metal oxide nanoparticles are widely used in industry, cosmetics, and biomedicine. However, the effects of exposure to these nanoparticles on the cardiovascular system remain unknown. The present study investigated the effects of nanosized TiO₂ and ZnO particles on the migration and adhesion of monocytes, which are essential processes in atherosclerogenesis, using an in vitro set-up of human umbilical vein endothelial cells (HUVECs) and human monocytic leukemia cells (THP-1). We also examined the effects of exposure to nanosized metal oxide particles on macrophage cholesterol uptake and foam cell formation. The 16-hour exposure to ZnO particles increased the level of monocyte chemotactic protein-1 (MCP-1) and induced the migration of THP-1 monocyte mediated by increased MCP-1. Exposure to ZnO particles also induced adhesion of THP-1 cells to HUVECs. Moreover, exposure to ZnO particles, but not TiO₂ particles, upregulated the expression of membrane scavenger receptors of modified LDL and increased cholesterol uptake in THP-1 monocytes/macrophages. In the present study, we found that exposure to ZnO particles increased macrophage cholesterol uptake, which was mediated by an upregulation of membrane scavenger receptors of modified LDL. These results suggest that nanosized ZnO particles could potentially enhance atherosclerogenesis and accelerate foam cell formation.     

乌梅水煎物对实验性溃疡性结肠炎小鼠的作用

目的探讨乌梅对溃疡性结肠炎(UC)小鼠结肠组织过氧化物歧化酶(SOD)活性及丙二醛(MDA)含量的影响。方法采用3%葡聚糖硫酸钠(DSS)溶液给小鼠自由饮水,建立小鼠溃疡性结肠炎模型,造模成功后,正常组、模型组给予0.33 ml生理盐水灌胃,乌梅低、中、高剂量组分别按设计剂量灌胃,每天1次,均连续给药10 d,期间进行隐血试验和观察粪便性状以及进行疾病活动度(DAI)评分;肉眼观察结合显微镜观察结肠组织形态学评分;测定病变结肠组织中SOD活性与MDA含量。结果发现乌梅中、高剂量组的小鼠无肉眼血便,可见松散大便。乌梅低、中、高剂量组的DAI均降低,中、高剂量组与模型组比较具有极显著性差异(P<0.01),在结肠黏膜病理改变中,乌梅高剂量组黏膜糜烂不严重,溃疡小,黏膜充血、水肿程度轻;在测定的SOD活力值中,中、高剂量组与模型组比较具有极显著性差异(P<0.01),均明显高于模型组。中剂量组与正常组比较具有显著性差异(P<0.05),高剂量组与正常组比较无显著性差异。测得MDA含量中,中、高剂量组与模型组比较具有极显著性差异(P<0.01),均明显低于模型组。结论提示自由基与UC的结肠组织损伤密切相关,参考UC的病理过程,乌梅对UC有明显治疗效果,并可能通过提高病变组织的SOD活性与降低MDA含量来发挥治疗作用。