Endothelial cells stimulate osteogenic differentiation of mesenchymal stem cells on calcium phosphate scaffolds

The interaction of mesenchymal stem cells (MSCs) with endothelium in vivo is significant for regenerative processes in organisms. To design concepts for tissue engineering for bone regeneration based on this interaction, the osteogenic differentiation of human bone marrow‐derived MSCs in a co‐culture with human dermal microvascular endothelial cells (HDMECs) was studied. The experiments were focussed on the regulation of MSCs in a co‐culture with HDMECs on different calcium phosphate scaffolds. Alkaline phosphatase (ALP) activity and mRNA expression of various osteogenic markers increased significantly when cells were co‐cultured on materials with calcium phosphate scaffolds compared to tissue culture polystyrene or when MSCs were cultured alone. In addition, it was observed that the expression of osteopontin and osteocalcin was highly sensitive to the substrate for cell adhesion. Whereas these late osteogenic markers were down‐regulated in co‐cultures on polystyrene, they were up‐regulated on calcium phosphate and moreover, were differentially expressed on the three calcium phosphate scaffolds tested. To enhance the osteogenic differentiation of MSCs in a co‐culture, direct cell‐cell interactions were required. Concerning molecular mechanisms in the interactions between both cell types, it was found that connexin 43 was expressed in contact sites and more apparently, endothelial cells grew over the MSCs, which facilitated direct cellular interactions mediated by various adhesion receptors. This study revealed significant findings for the design of implant materials suitable for regeneration of bone by stimulating the functional interaction of MSCs with endothelial cells. Copyright © 2012 John Wiley & Sons, Ltd.     

Endothelial cells and inflammation

Recent studies have brought remarkable progress in the understanding of cellular, subcellular, and molecular mechanisms of endothelial injury and responses to injury, both of which appear to be integral parts of the acute inflammatory response. The possible roles of endothelial surface enzymes in the inflammatory response are explored.     

内皮祖细胞与雌激素

背景：研究发现雌激素对血管内皮具有明显的保护作用,而内皮祖细胞作为内皮细胞的前体细胞参与内皮的修复。目的：总结内皮祖细胞生物特点及雌激素对内皮祖细胞作用的研究进展。方法：应用计算机检索PubMed数据库及CNKI数据库,在标题和摘要中以＂内皮祖细胞,雌激素＂或＂Endothelialprogenitorcells,estrogen＂为检索词进行检索。选择与内皮祖细胞生物学特点及雌激素对其作用研究相关的文献。结果与结论：内皮祖细胞存在于骨髓和外周血中,是具有增殖、迁移、黏附能力并分化为血管内皮细胞潜能的原始细胞,可作为未来治疗心血管疾病的重要的靶点。雌激素对内皮祖细胞有保护效应,能增强内皮祖细胞增殖、迁移、黏附等生物活性,同时还能延迟内皮祖细胞衰老、拮抗其凋亡。但雌激素影响内皮祖细胞生物活性的具体靶点及机制尚有待进一步研究。     

内皮祖细胞与雌激素

背景:研究发现雌激素对血管内皮具有明显的保护作用,而内皮祖细胞作为内皮细胞的前体细胞参与内皮的修复.目的:总结内皮祖细胞生物特点及雌激素对内皮祖细胞作用的研究进展.方法:应用计算机检索PubMed数据库及CNKI数据库,在标题和摘要中以"内皮祖细胞,雌激素"或"Endothelial progenitor cells,estrogen"为检索词进行检索.选择与内皮祖细胞生物学特点及雌激素对其作用研究相关的文献.结果与结论:内皮祖细胞存在于骨髓和外周血中,是具有增殖、迁移、黏附能力并分化为血管内皮细胞潜能的原始细胞,可作为未来治疗心血管疾病的重要的靶点.雌激素对内皮祖细胞有保护效应,能增强内皮祖细胞增殖、迁移、黏附等生物活性,同时还能延迟内皮祖细胞衰老、拮抗其凋亡.但雌激素影响内皮祖细胞生物活性的具体靶点及机制尚有待进一步研究.     

Endothelial cells and vascular hemostasis]

Procoagulant, anticoagulant, and fibrinolytic activities are associated with endothelial cells and involve the production, secretion, and receptor mediated binding of proteins involved in these processes. The procoagulant aspect of endothelial cells function involves the production and release of von Willebrand Factor(vWF), the production of tissue factor, and the presence of Factor IX/IXa receptors on the cell surface. Secretion of vWf will promote the initial steps in thrombus formation by supporting platelet-platelet interaction and platelet-subendothelial matrix adhesion. Tissue factor which is undetectable in resting cells appears after exposure to various cytokines and initiates factor VIIa activation of factors IX and X. Receptors of Factor IX/IXa are also present and mediate the assembly of the prothrombinase complex on the endothelial cell surface. The anticoagulant pathway involves the cell surface protein thrombomodulin, protein C and its cofactor protein S. Thrombomodulin binds thrombin which activates protein C which in the presence of protein S cleaves and inactivates Factors V and VIII. Inactivation of these two coagulation cofactors halts the coagulation. Finally, endothelial cells also play a pivotal role in the fibrinolytic system. Production and regulated secretion of tissue plasminogen activator creates a profibrinolytic state in the endothelial cell environment. In addition, receptors for plasminogen and urokinase are also present, constituting a cell surface mediated fibrinolytic pathway. Plasminogen activator inhibitor type I, the primary inhibitor of tPA, is also produced by endothelial cells. Thus endothelial cells can promote and inhibit fibrinolysis, depending on the prevailing environmental conditions.     

内皮祖细胞与缺血性脑卒中

背景:内皮祖细胞不仅能够早期预测血管损伤的程度,而且具有修复损伤的内皮细胞、促进新生血管形成管腔结构、参与神经再生的功能.内皮细胞移植已逐渐应用于血管相关性疾病的治疗.目的:文章综述了内皮祖细胞的生物学特性及在缺血件脑卒中领域的最新研究进展,为其临床应用提供理论支持.方法:以"endothelial progenitor cells: ischemic infarction:"为检索词检索Medline、HighWire Press数据库f2000 01/2009-12).纳入与血管新生,内皮和神经再生密切相关的研究,排除内容陈旧、重复性及缺乏可信度文章.结果与结论:计算机仞检得到126篇文献,根据纳入排除标准,对其中28篇文献进行分析.内皮祖细胞具有修复损伤的内皮细胞、延缓动脉粥样硬化进展、促进缺血组织新生血管形成等功能,它参与缺血件脑卒中后血管新生,防治支架术后血栓形成及再狭窄,预测脑缺血的发生及预后,在防治缺血性脑卒中方面具有广泛的应崩前景.     

Endothelial progenitor cells in health and atherosclerotic disease

Cardiovascular disease is associated with damage of the endothelial monolayer leading to endothelial dysfunction and atherosclerosis. A growing body of evidence suggests that circulating endothelial progenitor cells play an important role in endothelial cell regeneration. In this review we discuss the evolving role of stem- and progenitor cells in the maintenance of the vascular wall focusing on new pathophysiological concepts of endothelial cell regeneration. We discuss new insights into vascular stem cell biology derived from experimental and clinical studies.     

Endothelial cells and magnesium: implications in atherosclerosis

There is no doubt that the functional and structural integrity of the endothelium is critical in maintaining vascular homoeostasis and in preventing atherosclerosis. In the light of epidemiological and experimental studies, magnesium deficiency is emerging as an inducer of endothelial dysfunction. In particular, data on the effects of low extracellular magnesium on cultured endothelial cells reinforce the idea that correcting magnesium homoeostasis might be a helpful and inexpensive intervention to prevent and treat endothelial dysfunction and, consequently, atherosclerosis.     

Lymphocytes stimulate expression of 5-lipoxygenase and its activating protein in monocytes in vitro via granulocyte macrophage colony-stimulating factor and interleukin 3.

The aim of this study was to examine the role of lymphocytes in regulating expression of the 5-lipoxygenase pathway in monocytes. When monocytes were cultured over a period of days with lymphocytes, calcium ionophore-stimulated 5-lipoxygenase activity was enhanced. If lymphocytes alone were activated with lectins and their supernatants added to monocytes, stimulated 5-lipoxygenase activity was increased, whereas supernatants from lymphocytes cultured without lectins had no effect. Increased immunoreactive protein and mRNA for 5-lipoxygenase and 5-lipoxygenase activating protein were present in cells conditioned with lectin-activated lymphocyte supernatants. The effect of activated-lymphocyte supernatants could be mimicked by either GM-CSF or IL-3, but there was no additive effect with both cytokines. Both GM-CSF and IL-3 were present in the supernatant from lectin-activated lymphocytes at concentrations above their ED50, but were undetectable in the supernatant from nonactivated lymphocytes. The effect of lectin-activated lymphocyte supernatant could be inhibited by neutralizing antibodies to both cytokines, but not to either cytokine alone. We conclude that lymphocytes can regulate the expression of 5-lipoxygenase in monocytes, over a period of days, via the release of soluble factors, primarily GM-CSF and IL-3.     

Carbamylated proteins activate glomerular mesangial cells and stimulate collagen deposition

Carbamylated proteins formed in renal insufficiency from the spontaneous decomposition of urea exert a variety of metabolic effects. Here we examined the effects of carbamylated proteins on glomerular mesangial cells to determine whether urea retention in early renal insufficiency may itself promote glomerular sclerosis and hasten the progression to kidney failure. To this effect we carbamylated fetal bovine serum proteins in vitro and tested their effect on mesangial cell proliferation (by tritiated thymidine uptake), de novo protein synthesis (by tritiated leucine uptake), collagen I and collagen IV accumulation (by avidin-biotin enzyme immunoassay), and gelatinase levels in the medium (by zymography and quantitative fluorescence assay). Carbamylated fetal bovine serum at concentrations present in uremia increased tritiated thymidine incorporation by 50% without altering tritiated leucine incorporation, and it increased collagens I and IV in the monolayer by 150% to 300%. Gelatinase activity was unchanged. We conclude that carbamylated proteins can activate mesangial cells to a profibrogenic phenotype. From a clinical perspective, the carbamylation of proteins by elevated urea levels may accelerate the progression to kidney failure and thus set up a vicious cycle in which the nitrogen retention itself would cause further progression of fibrosis and deterioration of kidney function.     

Angiotensin II and vasopressin stimulate calcium-activated chloride conductance in rat mesangial cells

In an attempt to clarify the mechanisms by which angiotensin II (AII) and arginine vasopressin (AVP) regulate mesangial cell function, we examined the membrane potential change of mesangial cells and found that cells contracted and membrane potential depolarized in response to AII and AVP. The depolarization was associated with decreased input resistance. Ca ionophore A23187 caused similar mesangial cell contraction and depolarization. The reversal potential (Vr) of the depolarization response to AII and AVP was -29 +/- 3 and -25 +/- 7 mV (mean +/- SD), respectively. Not only the Vr of the AII-induced depolarization but also Vr of the Ca ionophore-induced response was dependent upon the extracellular Cl- concentration. Further, AII and AVP caused cell contraction and membrane depolarization in Ca++-free medium containing 0.5 mM EGTA. These data suggest the presence of Ca++ -activated Cl- channels in the mesangial cells and that AII and AVP increase Cl- permeability via an elevation of [Ca++]i released from the intracellular organellae.     

Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor.

Antigen-presenting, major histocompatibility complex (MHC) class II-rich dendritic cells are known to arise from bone marrow. However, marrow lacks mature dendritic cells, and substantial numbers of proliferating less-mature cells have yet to be identified. The methodology for inducing dendritic cell growth that was recently described for mouse blood now has been modified to MHC class II-negative precursors in marrow. A key step is to remove the majority of nonadherent, newly formed granulocytes by gentle washes during the first 2-4 d of culture. This leaves behind proliferating clusters that are loosely attached to a more firmly adherent "stroma." At days 4-6 the clusters can be dislodged, isolated by 1-g sedimentation, and upon reculture, large numbers of dendritic cells are released. The latter are readily identified on the basis of their distinct cell shape, ultrastructure, and repertoire of antigens, as detected with a panel of monoclonal antibodies. The dendritic cells express high levels of MHC class II products and act as powerful accessory cells for initiating the mixed leukocyte reaction. Neither the clusters nor mature dendritic cells are generated if macrophage colony-stimulating factor rather than granulocyte/macrophage colony-stimulating factor (GM-CSF) is applied. Therefore, GM-CSF generates all three lineages of myeloid cells (granulocytes, macrophages, and dendritic cells). Since > 5 x 10(6) dendritic cells develop in 1 wk from precursors within the large hind limb bones of a single animal, marrow progenitors can act as a major source of dendritic cells. This feature should prove useful for future molecular and clinical studies of this otherwise trace cell type.     

Pulmonary granulocyte kinetics in relation to endothelial and granulocyte activation

The aim of the study was to measure the peripheral blood levels of soluble E-selectin in patients with systemic inflammation and compare them with in vivo granulocyte activation, pulmonary intravascular granulocyte pooling, pulmonary extravascular granulocyte migration and 99mTc-diethylenetriaminepenta-acetic acid (DTPA) aerosol clearance, an index of lung injury. The level of soluble E-selectin was measured by capture ELISA. Granulocytes were labelled with 111In and 99mTc for quantification of pulmonary granulocyte kinetics. The pulmonary vascular granulocyte pool (PGP) was expressed as a fraction of the total blood granulocyte pool. Pulmonary granulocyte migration was quantified on 24-h images using the 111In signal. Granulocyte activation was quantified as the percentage of circulating cells showing shape change ('primed'). Lung injury was assessed from the clearance rate of inhaled 99mTc-DTPA aerosol. Eighteen patients with systemic inflammation were studied: five with inflammatory bowel disease, eight with systemic vasculitis, four with graft versus host disease and one with a recent renal transplant. The peripheral blood levels of soluble E-selectin were significantly elevated in patients with systemic inflammation. The level of soluble E-selectin showed a significant association with granulocyte migration (Spearman rank correlation coefficient, Rs=0.53; P<0.05) but not with PGP or with the percentage of cells showing shape change (P>0.05 for both). Granulocyte migration was bimodal: patients were therefore subdivided into 'migrators' and 'non-migrators'. Soluble E-selectin level, 99mTc-DTPA clearance and PGP, but not the percentage of cells showing shape change, were significantly higher in migrators than in non-migrators. We conclude that pulmonary intravascular granulocyte pooling is increased in the presence of increased numbers of circulating primed granulocytes but increased pooling does not by itself promote granulocyte migration into the lung interstitium. Insofar as an elevated level of E-selectin in peripheral blood reflects vascular endothelial activation, the data are consistent with the notion that pulmonary endothelial activation is required, in addition to granulocyte activation and an expanded PGP, for granulocyte migration into lung parenchyma and, therefore, for lung injury to occur.     

Cells in bone marrow and in T cell colonies grown from bone marrow can suppress generation of cytotoxic T lymphocytes directed against their self antigens

Both normal mouse bone marrow and cells from T cell-containing colonies grown in vitro from normal bone marrow contain cells which can specifically suppress the development of cytotoxic T lymphocytes capable of recognizing alloantigens on the bone marrow or colony cells. Suppression, as assessed by reduction in cytotoxic activity, is produced by adding bone marrow or colony cells to mixed lymphocyte reactions between lymph node responder cells and irradiated histoincompatible spleen stimulator cells. The cytotoxic activity is reduced if the added bone marrow or colony cells are syngeneic or semisyngeneic to the stimulator cells but not if they are allogeneic. Suppression results from a reduction in the number of cytotoxic lymphocyte precursor cells activated in the cultures. The suppressor cells in bone marrow are radiation sensitive and Thy-1 negative; those in colonies grown from bone marrow are radiation resistant and Thy-1 positive.     

Human cytomegalovirus reactivation in bone-marrow-derived granulocyte/monocyte progenitor cells and mature monocytes

Monocyte/granulocyte progenitor cells of the bone marrow are a major site of human cytomegalovirus (HCMV) latency. The mechanisms of establishment and maintenance of HCMV latency are still unknown. Reactivation of the latent virus in bone-marrow-derived progenitor cells has been demonstrated in vitro and suggested to occur also in vivo. Clinical studies have shown that reactivation is a rather frequent event not only in immunosuppressed but also in nonimmunosuppressed patients and in healthy blood donors. At least three independent mechanisms of virus reactivation are discussed: systemic inflammation connected with strong tumor necrosis factor alpha release; application of cAMP-elevating drugs, and highly stressful events associated with increased plasma catecholamine levels.     

Endothelial progenitor cells: new perspectives and applications in cardiovascular therapies

For over 10 years, bone marrow-derived endothelial progenitor cells (EPCs) have been studied as a novel biomarker to assess the severity of cardiovascular diseases, and as a potential new strategy in regenerative medicine. Cell-based therapy to stimulate postnatal vasculogenesis or to repair vascular integrity is being evaluated for cardiovascular diseases with excess morbidity and mortality, including ischemic heart disease, in-stent restenosis, pulmonary hypertension and peripheral arterial occlusive disease. Although clinical experience is still limited, observed effects appear modest compared with preclinical models. In this review, we will examine major hurdles to the effective use of EPCs, including our incomplete understanding of the characterization and dysfunctional phenotype of circulating EPCs in pathological conditions. Understanding the basic mechanisms of EPC dysfunction will be a prerequisite in enhancing their therapeutic potential.     

脐血内皮祖细胞的分离和培养

目的:研究从脐带血中分离内皮祖细胞的分离、培养及向内皮细胞方向诱导分化方法和条件。方法:从新鲜脐血中用密度梯度离心方法分离出单核细胞,在添加VEGF的M199培养液中培养,每隔3-4 d换一次液。培养16 d后,消化贴壁细胞,用DiI-ac-LDL及FITC-UEA-1对其进行免疫荧光及流式细胞仪分析。结果:培养3-4 d单核细胞开始贴壁,14 d左右开始出现索条状结构,免疫荧光鉴定显示贴壁细胞呈双荧光染色阳性,流式细胞仪分析显示70%贴壁细胞表达FITC-UEA-1, 85%贴壁细胞表达DiI-ac-LDL。结论:脐血中富含内皮祖细胞,在一定的培养条件下,可分化为内皮样细胞。     

Endothelial cells LEENE on noncoding RNAs in diabetic vasculopathy

Long noncoding RNAs (lncRNAs) have emerged as key mediators of regulated gene expression in diverse biologic contexts, including cardiovascular disease. In this issue of the JCI, Tang, Luo, and colleagues explored the contributions of lncRNAs in diabetic vasculopathy. The authors identified the lncRNA LEENE as a key mediator of angiogenesis and ischemic response. In a model of diabetic peripheral arterial disease, loss of LEENE led to impaired vascular perfusion, while its overexpression rescued the ischemic defect. The authors used unbiased chromatin affinity assays to decipher LEENE’s interactome and mode of action. These findings offer insights as to why patients with diabetes are uniquely susceptible to developing peripheral vascular disease and fill important gaps in our understanding of mechanisms that connect metabolic dysregulation with impaired angiogenesis.