The influence of anti-endothelial/antiphospholipid antibodies on fibrin formation and lysis on endothelial cells

The prothrombotic mechanisms associated with antiphospholipid antibodies remain incompletely defined. Antibody binding to endothelial cells in vitro is a feature of antiphospholipid antibody-positive sera. We hypothesised that impairment of endothelium-dependent fibrinolysis by antiphospholipid/anti-endothelial antibodies is a contributory factor in the pathogenesis of thrombosis. We also aimed to confirm the displacement of annexin-V from endothelial cells and enhanced fibrin formation. Binding of immunoglobulin (Ig) from antiphospholipid antibody-positive sera to endothelial cells was examined using a cell-based enzyme-linked immunosorbent assay. Effects on fibrin formation and lysis were examined on cultured endothelial cell monolayers. Plasminogen activator inhibitor-1 (PAI-1) was assayed in supernatants. We confirmed antibody binding to endothelial cells. With four of 14 antiphospholipid antibody-positive sera there was some prolongation of fibrin clot lysis time, consistent with impairment of endothelial fibrinolytic activity. Secretion of PAI-1 was significantly correlated with clot lysis time on endothelial cell monolayers incubated with antiphospholipid/anti-endothelial antibody-positive sera, but not with control sera. IgG from antiphospholipid antibody-positive sera had little effect on endothelial cell surface annexin-V expression. We conclude that impaired endothelial fibrinolysis is a potential prothrombotic mechanism in subjects with antiphospholipid antibodies. We were unable to confirm enhanced displacement of annexin-V from endothelium by antiphospholipid antibodies.     

Type I collagen fibril formation by human vascular endothelial cells in culture.

The formation of type I collagen fibrils by vascular human endothelial cells in culture was demonstrated by the indirect immunofluorescence method. The fibrillar structure was formed on the cell surface on the third day after subcultivation and had grown like a knitting ball of 0-3 microns in diameter and 0-200 microns in length on the seventh day. The fibril formation was stimulated by the addition of basic fibroblast growth factor, but completely blocked by the presence of beta-aminopropionitrile. The fibrils were eliminated by the treatment with clostridial collagenase or with 0.5% Triton X-100. The pathophysiological significance of type I collagen fibril formation by vascular endothelial cells in vascular diseases is also discussed.     

Collagen type 1 retards tube formation by human microvascular endothelial cells in a fibrin matrix

Angiogenesis, or the formation of new microvessels, is often encountered in pathological situations. A fibrinous exudate can often act as a temporary matrix for the ingrowth of these new microvessels. This matrix consists mainly of fibrin, but is mingled with other plasma components and interstitial collagen fibres. In vitro, capillary-like tube formation can be mimicked by exposing human microvascular endothelial cells (hMVECs), seeded on top of a three-dimensional fibrin matrix, to an angiogenic growth factor (e.g. fibroblast growth factor (FGF)-2) and the cytokine tumour necrosis factor (TNF)-. Plasmin activity is required in this process. We investigated whether the angiogenic potential of hMVECs was altered by the presence of collagen. The addition of type I collagen to fibrin matrices dose-dependently inhibited tube-formation. Tube-formation in these fibrin/collagen matrices by hMVECs required matrix metalloprotease (MMP) activity, as well as plasmin activity. On a pure collagen type I matrix, hMVECs were not able to form tube-like structures in the matrix but formed sprouts. This sprouting required MMP activity and was, in contrast to the tube-like structures in a fibrin matrix, not influenced by hypoxia. These data indicate that the interaction between endothelial cells and different matrix components is of importance for the angiogenic potential of these cells.     

Regulation of adipogenesis and osteogenesis in mesenchymal stem cells by vascular endothelial growth factor A

Understanding the mechanisms by which bone marrow mesenchymal stem cells (BMSCs) differentiate into bone‐forming osteoblasts and marrow adipocytes is crucial to develop strategies for the treatment of several bone diseases. Age‐related bone loss resulting in osteopenia and osteoporosis has been associated with reduced numbers of osteoblasts and increased numbers of adipocytes, likely originating from differentiation defects in BMSCs. Although many factors involved in the complex regulation of osteoblast and adipocyte cell lineages have previously been identified, their functional interactions in the context of BMSC differentiation and maintenance of bone homeostasis during ageing are unknown. Recent discoveries have provided important new insights into the mechanisms by which the nuclear envelope protein lamin A and vascular endothelial growth factor A (VEGF) mutually control BMSC fate. Particularly interesting is the finding that VEGF in this context functions as an intracellular protein, unaffected by neutralizing antibodies, and not as a secreted growth factor. These insights may not only facilitate the identification of new targets for treating bone diseases but also lead to improved design of tissue engineering approaches aimed at stimulating bone regeneration and repair.     

Mechanisms of vasculogenesis in 3D fibrin matrices mediated by the interaction of adipose-derived stem cells and endothelial cells

Vascularization of tissue-engineered constructs is essential to provide sufficient nutrient supply and hemostasis after implantation into target sites. Co-cultures of adipose-derived stem cells (ASC) with outgrowth endothelial cells (OEC) in fibrin gels were shown to provide an effective possibility to induce vasculogenesis in vitro. However, the mechanisms of the interaction between these two cell types remain unclear so far. The aim of this study was to evaluate differences of direct and indirect stimulation of ASC-induced vasculogenesis, the influence of ASC on network stabilization and molecular mechanisms involved in vascular structure formation. Endothelial cells (EC) were embedded in fibrin gels either containing non-coated or ASC-coated microcarrier beads as well as ASC alone. Moreover, EC-seeded constructs incubated with ASC-conditioned medium were used in addition to constructs with ASC seeded on top. Vascular network formation was visualized by green fluorescent protein expressing cells or immunostaining for CD31 and quantified. RT-qPCR of cells derived from co-cultures in fibrin was performed to evaluate changes in the expression of EC marker genes during the first week of culture. Moreover, angiogenesis-related protein levels were measured by performing angiogenesis proteome profiler arrays. The results demonstrate that proximity of endothelial cells and ASC is required for network formation and ASC stabilize EC networks by developing pericyte characteristics. We further showed that ASC induce controlled vessel growth by secreting pro-angiogenic and regulatory proteins. This study reveals angiogenic protein profiles involved in EC/ASC interactions in fibrin matrices and confirms the usability of OEC/ASC co-cultures for autologous vascular tissue engineering.     

Equal modulation of endothelial cell function by four distinct tissue-specific mesenchymal stem cells

Mesenchymal stem cells (MSCs) can generate multiple end-stage mesenchymal cell types and constitute a promising population of cells for regenerative therapies. Additionally, there is increasing evidence supporting other trophic activities of MSCs, including the ability to enable formation of vasculature in vivo. Although MSCs were originally isolated from the bone marrow, the presence of these cells in the stromal vascular fraction of multiple adult tissues has been recently recognized. However, it is unknown whether the capacity to modulate vasculogenesis is ubiquitous to all MSCs regardless of their tissue of origin. Here, we demonstrated that tissue-resident MSCs isolated from four distinct tissues have equal capacity to modulate endothelial cell function, including formation of vascular networks in vivo. MSCs were isolated from four murine tissues, including bone marrow, white adipose tissue, skeletal muscle, and myocardium. In culture, all four MSC populations secreted a plethora of pro-angiogenic factors that unequivocally induced proliferation, migration, and tube formation of endothelial colony-forming cells (ECFCs). In vivo, co-implantation of MSCs with ECFCs into mice generated an extensive network of blood vessels with ECFCs specifically lining the lumens and MSCs occupying perivascular positions. Importantly, there were no differences among all four MSCs evaluated. Our studies suggest that the capacity to modulate the formation of vasculature is a ubiquitous property of all MSCs, irrespective of their original anatomical location. These results validate multiple tissues as potential sources of MSCs for future cell-based vascular therapies.     

丝素蛋白/壳聚糖生物支架复合骨髓间质干细胞修复老年兔软骨缺损

目的 探讨丝素蛋白(SF)/壳聚糖(CS)生物支架复合诱导后骨髓间质干细胞(BMSCs)修复老年兔膝关节软骨缺损的可行性. 方法 分离培养及诱导BMSCs,将成功诱导后的BMSCs接种在SF-CS生物支架上构成修复体.54只16～18月龄兔,随机分为支架修复体组、单纯支架组和对照组,每组18只.采用右膝关节制备软骨缺损模型并植入支架.术后4、8、12周取材进行大体观察,组织学染色和改良Wakitani法组织学评分. 结果 SF-CS支架为相通性好的多孔结构,孔径平均151.72 μm,孔隙率为(92.72±4.78)％,吸水膨胀率为(141.10±6.87)％.BMSCs诱导后在SF-CS支架上生长良好,增殖活跃.12周时,支架修复体组软骨缺损基本修复,Ⅱ型胶原明显的阳性反应,生物材料基本吸收;单纯支架组以纤维样组织修复为主,Ⅱ型胶原阳性反应弱,未见支架残留;空白对照组修复不良;改良Wakitani评分显示支架修复体组优于单纯支架组和对照组(P＜0.05).结论 SF-CS生物支架可以作为BMSCs载体修复老年兔膝关节软骨缺损.     

CD13/APN regulates endothelial invasion and filopodia formation

CD13/aminopeptidase N is a transmembrane peptidase that is induced in the vasculature of solid tumors and is a potent angiogenic regulator. Here, we demonstrate that CD13 controls endothelial cell invasion in response to the serum peptide bradykinin by facilitating signal transduction at the level of the plasma membrane. Inhibition of CD13 abrogates bradykinin B(2) receptor internalization, leading to the attenuation of downstream events such as bradykinin-induced activation of Cdc42 and filopodia formation, and thus affects endothelial cell motility. Investigation into mechanisms underlying this block led us to focus on B(2)R internalization via membrane-dependent mechanisms. Membrane disruption by depletion of cholesterol or trypsinization halts B(2)R internalization, invasion, and filopodia formation, which can be recovered with addition of cholesterol. However, this functional recovery is severely impaired in the presence of CD13 antagonists, and the distribution of membrane proteins is disordered in treated cells, suggesting a role for CD13 in plasma membrane protein organization. Finally, exogenous expression of wild-type but not mutant CD13 further alters protein distribution, suggesting peptidase activity is required for CD13's regulatory activity. Therefore, CD13 functions as a novel modulator of signal transduction and cell motility via its influence on specific plasma membrane organization, thus regulating angiogenesis.     

Establishment of a three-dimensional co-culture system by porcine hepatocytes and bone marrow mesenchymal stem cells in vitro

Aim: The application of porcine hepatocytes in liver support systems has been hampered by the short‐term survival. Co‐cultivation of hepatocytes with non‐parenchymal cells may be beneficial for optimizing cell functions via heterotypic interactions. In this study, we present a new cultivation system of porcine hepatocytes and mesenchymal stem cells (MSCs) in a randomly distributed co‐culture manner. Methods: Mononuclear cells were isolated from bone marrow aspirate of swines (n = 3) by density gradient centrifugation. MSCs were characterized by flow cytometry with CD29, CD44, CD45 and CD90, respectively. Then freshly isolated hepatocytes were simultaneously inoculated with MSCs in a hepatocyte dominant manner. The morphological and functional changes of heterotypic interactions were characterized. Results: Ninety percent MSCs of passage 3 were positive for CD29, CD44 and CD90, but negative for CD45. A rapid attachment and self‐organization of three‐dimensional hepatocyte aggregates were encouraged. The cell ultrastructure indicating heterotypic junctions remained similar to that of hepatocytes in vivo. Fluorescence microscopy further verified that MSCs served as a feeder layer for hepatocyte aggregates. Hepatocyte performance levels such as albumin secretion, urea synthesis and CYP3A1 induction were all significantly enhanced in co‐culture group compared with hepatocyte homo‐culture (P < 0.05). The best hepatic function levels were achieved on day 2 and moderately decreased in the following co‐culture days. Moreover, the cell cycle of hepatocytes manifested the same trend in parallel to the enhancement of hepatocyte functionality. Conclusions: A three‐dimensional co‐culture system by porcine hepatocytes and bone marrow MSCs was for the first time established in vitro. Enhanced liver‐specific functions make such a co‐culture system a promising tool for tissue engineering, cell biology, and bioartificial liver devices.     

心肌联合注射纤维蛋白封闭剂在细胞移植中应用的研究

目的:探讨心肌注射骨髓间充质干细胞(mesenchymal stemcells,MSCs)联合应用纤维蛋白封闭剂(FibrinSealant,FS),对细胞滞留量的影响。方法:(1)提取、分离、培养、扩增小型猪骨髓MSCs备用;(2)小型猪随机分为2组,成功建立急性心肌梗塞模型,然后分为单纯MSCs移植组和MSCs联合纤维蛋白封闭剂移植组,对比移植6周后细胞的滞留量及新生血管的密度,超声学评价心脏功能。结果:6周后MSCs FS组荧光标记的MSCs数量、新生血管密度均较MSCs组显著增多(P<0.01),MSCs FS组左室短轴缩短率(FS%)、心肌射血分数(EF%)较MSCs组显著提高(P<0.05,<0.01)。结论:经心外膜心肌注射MSCs联合纤维蛋白封闭剂较单纯心肌注射MSCs能够明显提高移植细胞的滞留存活数量,增加新生血管密度,改善心肌收缩功能。     

The interactions between brain microvascular endothelial cells and mesenchymal stem cells under hypoxic conditions

The purpose of the present study was to investigate the interactions between brain microvascular endothelial cells (BMEC) and mesenchymal stem cells (MSC) under hypoxic conditions. Primary cultured human bone marrow MSC and rat BMEC were isolated, cultured and identified. Vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) were detected in the conditioned media of BMEC and MSC under normal and hypoxic conditions using ELISA. MSC differentiation was analyzed using flow cytometry and fluorescence immunocytochemistry. Transendothelial electrical resistance (TEER) techniques were employed to measure changes in permeability across the BMEC monolayer. Under hypoxic conditions, the concentration of VEGF and MMP-9 in the conditioned media increased significantly, with greater levels in the MSC than the BMEC media. Primary MSC did not express vWF and Flk-1. MSC were co-cultured with BMEC under hypoxic conditions 5 days later. MSC expressing Flk-1 accounted for 23.64+/-2.50% (n=6, P<0.001) of the total number of cells. Interestingly, some Flk-1 positive cells began to coexpress vWF simultaneously. Under hypoxic conditions, MSC conditioned media significantly enhanced the proliferation and migration of BMEC. In addition, MSC decreased the TEER of the BMEC monolayer (lowest values: 50.5+/-2.6% of the original), which could partially be inhibited by both anti-VEGF antibody and MMP-9 inhibitor. These data indicate that under hypoxic conditions BMEC induce MSC to differentiate into endothelial cells, and MSC enhance the proliferation and migration of BMEC through paracrine functions, while simultaneously increasing the permeability of the BMEC monolayer.     

Influence of fibrin structure on the formation and maintenance of capillary-like tubules by human microvascular endothelial cells

Fibrin is a temporary matrix which not only covers a wound, but also provides a structure for invading cells during healing. Changes in the polymerization conditions before gelation of the clot affect the structure of fibrin and thus might influence the interaction with invading cells. Therefore we tested whether changes in the fibrin structure influence the formation of capillary-like tubular structures by human microvascular endothelial cells (hMVEC) in an in vitro angiogenesis model. Opaque [¹²⁵I]fibrin structures prepared at pH 7.0, fibrin matrices at pH 7.4 and transparent [¹²⁵I]fibrin structures prepared at pH 7.8 were neutralized (pH 7.4) before seeding hMVEC on top of them in confluent density. Endothelial cells were stimulated with a growth factor [basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF)₁₆₅] and a cytokine [tumor necrosis factor (TNF)-α] to induce the u-PA/u-PA receptor-dependent formation of capillary-like tubular structures. The formation of these structures was quantified by determining the length of the invasive structures by image analysis and by measuring the accompanying [¹²⁵I]fibrin degradation. Ingrowth of tubular structures proceeded at a faster rate in opaque matrices consisting of thick fibrin fibers as compared to transparent gels with fine fibrin fibers. The more rapid ingrowth of tubular structures in opaque fibrin gels induced by bFGF/TNF-α or VEGF₁₆₅/TNF-α was accompanied by a larger extent of fibrin degradation. Both processes were inhibited by aprotinin and ∈-aminocaproic acid indicating the involvement of plasmin. They were also inhibited by anti-u-PA or anti-u-PA receptor IgG, but not by anti-t-PA IgG, suggesting the involvement of cell-bound u-PA activity. However, in the opaque fibrin gels, the tubular structures dissolved upon prolonged incubation due to excessive fibrin degradation. Simulation of hMVEC with bFGF alone did not induce tubular structures, but ca used a high degree of t-PA- and plasmin-dependent fibrin lysis, and, after several days, a partial detachment of sheets of cells. Gradual inhibition of the excessive fibrin degradation by a series of aprotinin concentrations did not lead to tube formation in bFGF-treated cells. These data indicate that the formation and stability of tubular structures by hMVEC in fibrin is accompanied by controlled fibrinolysis and depends critically not only on cell-bound u-PA-dependent plasminogen activation, but also on the fibrin structure. Because the fibrin structure is largely influenced by the conditions in which fibrin has been polymerized, these conditions may have considerable impact on angiogenesis during wound healing and vascularization of tumour stroma. This revised version was published online in August 2006 with corrections to the Cover Date.     

Immunomodulation by mesenchymal stem cells and clinical experience

Mesenchymal stem cells (MSCs) from adult marrow can differentiate in vitro and in vivo into various cell types, such as bone, fat and cartilage. MSCs preferentially home to damaged tissue and may have therapeutic potential. In vitro data suggest that MSCs have low inherent immunogenicity as they induce little, if any, proliferation of allogeneic lymphocytes. Instead, MSCs appear to be immunosuppressive in vitro. They inhibit T-cell proliferation to alloantigens and mitogens and prevent the development of cytotoxic T-cells. In vivo, MSCs prolong skin allograft survival and have several immunomodulatory effects, which are presented and discussed in the present study. Possible clinical applications include therapy-resistant severe acute graft-versus-host disease, tissue repair, treatment of rejection of organ allografts and autoimmune disorders.     

血管内皮生长因子基因转染骨髓间充质干细胞移植促进缺血心肌血管生成的研究

目的研究血管内皮生长因子(vascularendothelial growth factor,VEGF)基因转染骨髓间充质干细胞(mesenchymalstem cells,MSCs)移植对缺血心肌的血管生成作用。方法于2004年5月至2005年8月取第四军医大学西京医院分离、培养Wistar大鼠的MSCs,用真核表达载体pcDNA3.1(-)/hVEGF165转染MSCs。45只近交系Wistar大鼠随机均分为转染组(MSCs/VEGF组)、对照组(MSCs组)、无血清培养基组(DMEM组),结扎前降支建立急性心肌梗死模型后在梗死区边缘区行5×106细胞移植,DMEM组行等量培养基注射。细胞移植前行CM-DiI标记。移植1个月后行心脏B超测量射血分数值,组织化学染色评价新生血管密度。结果培养的MSCs呈典型贴壁生长成纤维样外观,pcDNA3.1(-)/hVEGF165能有效转染大鼠MSCs,移植1个月后MSCs/VEGF组较其余各组左室射血分数(LVEF),再生血管密度明显增加,差异均有显著性(P<0.01)。结论VEGF基因转染MSCs移植能显著促进缺血心肌血管再生,进而改善心脏功能。     

兔骨髓间充质干细胞体外成内皮细胞能力的研究

目的　探讨骨髓间充质干细胞体外扩增及其向内皮细胞定向分化能力。方法　无菌条件下采集兔骨髓 ,肝素抗凝 ,经淋巴细胞分层液密度梯度离心分离骨髓单个核细胞 ,通过贴壁培养法获得MSC ,然后在内皮细胞生长环境中进行诱导分化 ,最后对扩增的细胞特征进行鉴定 ,同时初步研究其体外成血管特性。结果　MSC每扩增一代 ,细胞数量增加 5～ 8倍 ,7.6 5× 10 3 个原代MSCs体外扩增 3代即获得 1.2 6× 10 8个细胞。在 70 %～80 %融合时呈现“铺路石”样形态 ,细胞免疫组化证实扩增细胞表达CD31和vonWillebrandfactor(vWF) ,具有吞噬ac LDL的功能 ,且扩增细胞在体外参与网状血管样结构形成。结论　MSC扩增能力强 ,在体外能诱导其向内皮细胞方向分化。     

血小板衍生生长因子−C对体外培养血管内皮细胞及间充质干细胞的影响

目的 观察血小板衍生生长因子?C（PDGF-C）对体外培养的血管内皮细胞（VECs）及间充质干细胞（MSCs）的影响。方法　采用培养的大鼠主动脉内皮细胞和MSCs,应用细胞直接计数法、噻唑蓝比色法（MTT法）评价细胞的增殖情况,流式细胞术检测细胞周期,利用Transwell细胞迁移实验和细胞划痕实验测定细胞迁移能力。结果　PDGF-C能明显刺激VECs和MSCs的增殖,增加两种细胞S期细胞的比例,对VECs增殖呈剂量依赖关系,对MSCs的促增殖作用在20μg/L达高峰。同时,PDGF-C促进VECs和MSCs的迁移能力,对MSCs迁移作用强于VECs。结论　PDGF-C可促进培养的VECs、MSCs的增殖与迁移能力。     

兔骨髓间充质干细胞的鉴定及向血管内皮细胞的分化

目的探要诱导骨髓间充质干细胞(BMSCs)分化为血管内皮细胞(VECs)可行性,并比较传代对诱导率的影响。方法采用密度梯度离心法分离BMSCs,体外培养扩增并做形态学、细胞表型和成骨成脂诱导鉴定。细胞纯化后行内皮化诱导,诱导组分A、B两组,C组为对照组,A组于诱导第15天传代,B组常规诱导,24 d后终止培养,从形态学、CD31流式分析及一氧化氮(NO)分泌量检测三方面进行鉴定和比较。结果经鉴定,分离出来的细胞为纯度较高分化能力良好的BMSCs,内皮化诱导后,细胞呈现典型的内皮细胞形态,CD31和NO分泌量的检测证实诱导后的细胞为具有活性的内皮细胞,并且A组的诱导率高于B组。结论 BMSCs可以被诱导为VECs,诱导过程中传代可以提高诱导率。     

CD48 on hematopoietic progenitors regulates stem cells and suppresses tumor formation

The proliferation and differentiation of adult stem cells is balanced to ensure adequate generation of differentiated cells, stem cell homeostasis, and guard against malignant transformation. CD48 is broadly expressed on hematopoietic cells but excluded from quiescent long-term murine HSCs. Through its interactions with CD244 on progenitor cells, it influences HSC function by altering the BM cytokine milieu, particularly IFNγ. In CD48-null mice, the resultant misregulation of cytokine signaling produces a more quiescent HSC, a disproportionate number of short-term progenitors, and hyperactivation of Pak1, leading to hematologic malignancies similar to those found in patients with X-linked lymphoproliferative disease. CD48 plays a vital role as an environmental sensor for regulating HSC and progenitor cell numbers and inhibiting tumor development.