Transmission electron microscopy procedure for endothelial cell cultures with angiogenesis

Determination of the structural characteristics of angiogenesis requires a procedure protective of the forming vascular fibers and the endothelial cell monolayer exhibiting cord formation. This report describes in situ fixation of angiogenic cultures in 96-well plates and the subsequent double embedding processing for electron microscopy. Cross sections of the monolayer are obtained without damage of the incipient capillaries.     

人外周血内皮祖细胞的分离、培养和扩增

目的:改进从人外周血中分离、培养和体外扩增血管内皮祖细胞(EPC)的方法。方法:采用不同淋巴细胞分离液,用密度梯度离心法从外周血分离EPC;用CD34免疫磁性活化细胞分选系统(MACS)分离CD34 细胞;分别培养在包被和不包被有人纤维连接蛋白(HFN)的培养板内;采用细胞免疫化学法检测内皮细胞表面标志CD31、CD34和vWF的表达。结果:从成人外周血可分离获得EPC;不同的分离条件可影响获得EPC的数量和质量,HFN对EPC的生长有促进作用。结论:进一步改进从人外周血分离获取EPC并行体外扩增的方法,为EPC的研究奠定了基础。     

Crosslinked gelatin beads: A culture method for the study of cell adhesion and invasion

Summary A method is described for culturing invasive cell lines on crosslinked gelatin beads and preparing them for immunocytochemical and morphological observation. Very invasive cells such as Rous sarcoma virus-transformed chicken embryo fibroblasts, and human melanoma LOX and RPMI7951 and breast carcinoma MDA-MB-231 cells will actively degrade this matrix, extending cellular protrusions, called invadopodia, into the sites of degradation. Normal chicken embryo fibroblasts and other non-invasive cell lines do not disrupt the surface of these beads and do not form invadopodia. Invadopodia extending into the bead can be visualized by electron microscopy. Cellular removal of fluorescent fibronectin that has been covalently coupled to the bead surface can be monitored using fluorescence microscopy of frozen-thin-sections. In double label experiments, immunocytochemistry is used to localize antigens in invadopodia at sites of membrane invasion. The materials for bead preparation are inexpensive, and this method has the advantage that many cell types will attach and spread readily on the beads, while only highly invasive cells will invade into the bead.     

Hypoxia-induced angiogenesis is increased by the controlled release of deferoxiamine from gelatin hydrogels

The objective of this study is to design biodegradable hydrogels for the controlled release of deferoxiamine (DFO) and evaluate their biological activity. When the DFO was added to human umbilical vein endothelial cells cultured in 5.0% O₂, the level of hypoxia-inducible factor-1α and vascular endothelial growth factor significantly increased compared with that without DFO. The expression of angiogenesis-related genes was accordingly increased by the DFO addition. An aqueous solution of mixed gelatin and DFO was freeze-dried, and dehydrothermally treated at 140 °C for 24 h to prepare a gelatin hydrogel incorporating DFO. In the release test with phosphate-buffered saline solution (PBS) at 37 °C, an initial DFO release of 60% was observed, followed by no release. When placed in PBS containing collagenase, the hydrogel was enzymatically degraded with time, and consequently released DFO in a degradation-dependent manner. After the hydrogel incorporating DFO was injected intramuscularly into a mouse model of hind limb ischemia, the number of new blood vessels formed was significantly higher than that with free DFO and DFO-free hydrogel. It is concluded that the DFO-containing hydrogel shows promising for inducing angiogenesis locally.     

Enhanced angiogenesis of growth factor-free porous biodegradable adhesive made with hexanoyl group-modified gelatin

The bonding behavior of hexanoyl (Hx: C₆) group-modified alkaline-treated gelatin (HxAlGltn) porous films ((P)HxAlGltn) on the porcine intestine was evaluated. (P)HxAlGltns with various porosities were prepared by the salt-leaching method for various solid–liquid ratios. (P)HxAlGltns bonded more strongly to porcine intestine surfaces than did porous AlGltn films ((P)AlGltns). L929 cells cultured on (P)HxAlGltns showed adhesivity than cells cultured on (P)AlGltns. Faster tissue infiltration and a shorter degradation time of highly porous (P)HxAlGltns were observed after implantation in rat subcutaneous tissues. The angiogenic markers CD34 and α-SMA were highly expressed around (P)HxAlGltns that had high porosity. These results indicated that highly porous (P)HxAlGltns have advantages with respect to not only bonding strength on wet soft tissues, but also angiogenesis.     

The 3D printing of gelatin methacrylamide cell-laden tissue-engineered constructs with high cell viability

In the present study, we report on the combined efforts of material chemistry, engineering and biology as a systemic approach for the fabrication of high viability 3D printed macroporous gelatin methacrylamide constructs. First, we propose the use and optimization of VA-086 as a photo-initiator with enhanced biocompatibility compared to the conventional Irgacure 2959. Second, a parametric study on the printing of gelatins was performed in order to characterize and compare construct architectures. Hereby, the influence of the hydrogel building block concentration, the printing temperature, the printing pressure, the printing speed, and the cell density were analyzed in depth. As a result, scaffolds could be designed having a 100% interconnected pore network in the gelatin concentration range of 10–20 w/v%. In the last part, the fabrication of cell-laden scaffolds was studied, whereby the application for tissue engineering was tested by encapsulation of the hepatocarcinoma cell line (HepG2). Printing pressure and needle shape was revealed to impact the overall cell viability. Mechanically stable cell-laden gelatin methacrylamide scaffolds with high cell viability (>97%) could be printed.     

大鼠胰岛微血管内皮细胞分离、纯化与培养的改进

目的：建立稳定可靠的大鼠胰岛微血管内皮细胞分离培养方法。方法：分离纯化大鼠胰岛后进行内皮细胞选择性培养，使用UEA-1包被的免疫磁珠对胰岛微血管内皮细胞进行纯化。免疫荧光法检测经典的内皮细胞标志物第Ⅷ因子相关抗原（vWF）和CD34的表达和吞噬Dil标记的乙酰化低密度脂蛋白（Dil-Ac-LDL）能力。结果：胰岛培养4-5 d后，可见内皮细胞从贴壁的胰岛内爬出，通过UEA-1包被的免疫磁珠筛选出胰岛微血管内皮细胞接种后24 h细胞开始贴壁。该细胞具有单层生长、接触抑制的特性。大鼠胰岛微血管内皮细胞表达vWF和CD34，可摄取Dil-Ac-LDL。结论：本研究方法是一种较为高效的分离、纯化和培养大鼠胰岛微血管内皮细胞的方法。     

内皮祖细胞在体外培养成血管样结构的初步观察

目的：探索体外培养脐血、外周血内皮祖细胞（EPCs）的方法，观察其形成血管样结构的可能性及条件。 方法： 采用贴壁选择法培养人脐血及兔外周血内皮祖细胞，光镜下观察细胞形态，用荧光显微镜、流式细胞仪分析贴壁细胞CD34、VEGFR-2、AC133、血管内皮钙粘素（VE-cadherin）的表达，DiI-ac-LDL 吞噬试验及Ⅷ因子免疫组化证实细胞属性。 结果： 体外成功培养出人脐血及兔外周血内皮祖细胞，形成条索状、管状结构，兔外周血EPCs分化较成熟，形成典型铺路石形状及血管样结构。 结论： 脐血、兔外周血内皮祖细胞可在体外培养成功并表现成血管倾向，可能是血管组织工程的潜在资源。     

虎杖甙对白细胞-内皮细胞粘附作用的影响

目的探讨虎杖甙改善微循环的作用机理。方法在细胞液流室中,观察虎杖甙（ＰＤ）对血液中性粒细胞与培养的血管内皮细胞粘附性的影响。结果在正常情况下,随着切应力增大,白细胞与内皮细胞粘附率逐渐下降,但ＩＬ－８处理组和失血性休克组的白细胞,其与内皮的粘附率都明显高于对照组。而这两组的白细胞如果事先与ＰＤ温育３０ｍｉｎ后,再与内皮细胞作用,则白细胞与内皮的粘附率明显下降到接近正常对照水平。如果中性粒细胞与内皮细胞先粘附并以一定切应力灌流后再加入ＰＤ溶液孵育,也发现ＰＤ能显著降低白细胞与内皮的粘附率。结论ＰＤ能通过直接降低白细胞对血管内皮的粘附性而改善微循环。     

双特异抗体增效内皮祖细胞移植促进心肌血管新生

目的探讨在双特异抗体(BiAb)的辅助下,内皮祖细胞(EPCs)移植可否更好的定向归巢大鼠缺血心肌促进血管新生。方法体外分离培养鉴定SD大鼠骨髓源性内皮祖细胞;开胸结扎SD大鼠冠状动脉左前降支制备心肌梗死模型;以anti-CD34(能识别内皮祖细胞)和抗肌凝蛋白轻链抗体(AMLCA)(能特异性结合缺血心肌)2种抗体,化学交联法制备BiAb(CD34×AMLCA)。将此BiAb与EPCs经尾静脉输入心肌梗死大鼠(EPCs+BiAb组),另设单纯EPCs移植组、单纯BiAb组、对照组。细胞移植35 d后M型超声心动图检测大鼠左室收缩功能,免疫组织化学法行5-Brdu及VIII因子检测,实时荧光定量PCR及Western blot检测大鼠心肌VEGF mRNA与蛋白表达。结果与其余组相比,EPCs+BiAb组射血分数及短轴缩短率增加,心梗区周围5-BrdU阳性细胞数及微血管密度增加,心肌VEGF mRNA与蛋白表达增加(P<0.05)。结论 CD34×AMLCA双特异抗体可增效大鼠骨髓源性内皮祖细胞定向归巢到大鼠缺血心肌,改善心功能,更好的促进血管新生。     

Culturing endothelial cells of microvascular origin

There is considerable divergence of opinion on the best methods for the isolation and in vitro culture of microvascular endothelium. Reports have either only described the isolation without mentioning culture conditions or have not fully defined the cell population being cultured. Even at the close of the 20th Century, the isolation and in vitro culture of endothelial cells of microvasculature origin still proves to be technically difficult and many questions remain. These questions need to be addressed by improvements to current methods of isolation and culture of Endothelial cells. A number of more 'high-tech' approaches to this isolation are being explored currently. Use of a more definitive panel of antibodies for immunocytochemical characterization, should enable a more confident characterization of the endothelial cell preparations cultured in vitro. Cell adhesion molecules such as ELAM and VCAM can be used to assist in determining cell population purity.     

Combinatorial approach for improving the outcome of angiogenic therapy in ischemic tissues

Two major populations of endothelial progenitor cells (EPC), namely endothelial colony forming cells (ECFC, or late outgrowth EPC) and circulating angiogenic cells (CAC, or early outgrowth EPC) have been reported to play important roles in vasculogenesis in numerous pathological conditions. However, the poor retention of cells into the ischemic tissue and neovessel fragility are two major flaws that need to be overcome for successful angiogenic therapy. The objective of this study was to explore and exploit the functional properties of EPC populations in order to increase the effectiveness of post-ischemic cell therapy. The results indicate different, still complementary, effects of the two EPC populations on adherence and proliferation of vascular endothelial cells. Matrigel plug assay and mouse hind limb ischemia model showed that concomitant administration of CAC-secreted factors and ECFC resulted in three-fold increase in local cell retention and improved muscle perfusion, vessel maturation and hind limb regeneration, in comparison to either treatment alone. By concluding, factors secreted by CAC co-administered at the time of ECFC transplantation improve tissue regeneration and vascular repair through stabilization of newly-derived blood vessels.     

Paracrine mechanisms of endothelial progenitor cells in vascular repair

Endothelial progenitor cells (EPCs) play an important role in repairing damaged blood vessels and promoting neovascularization. However, the specific mechanism of EPCs promoting vascular repair is still unclear. Currently, there are two different views on the repair of damaged vessels by EPCs, one is that EPCs can directly differentiate into endothelial cells (ECs) and integrate into injured vessels, the other is that EPCs act on cells and blood vessels by releasing paracrine substances. But more evidence now supports the latter. Therefore, the paracrine mechanisms of EPCs are worth further study. This review describes the substances secreted by EPCs, some applications based on paracrine effects of EPCs, and the studies of paracrine mechanisms in cardiovascular diseases--all of these are to support the view that EPCs repair blood vessels through paracrine effects rather than integrating directly into damaged vessels.     

Enhancing microvascular formation and vessel maturation through temporal control over multiple pro-angiogenic and pro-maturation factors

Therapeutic stimulation of angiogenesis to re-establish blood flow in ischemic tissues offers great promise as a treatment for patients suffering from cardiovascular disease or trauma. Since angiogenesis is a complex, multi-step process, different signals may need to be delivered at appropriate times in order to promote a robust and mature vasculature. The effects of temporally regulated presentation of pro-angiogenic and pro-maturation factors were investigated in vitro and in vivo in this study. Pro-angiogenic factors vascular endothelial growth factor (VEGF) and angiopoietin 2 (Ang2) cooperatively promoted endothelial sprouting and pericyte detachment in a three-dimensional in vitro EC-pericyte co-culture model. Pro-maturation factors platelet-derived growth factor B (PDGF) and angiopoietin 1 (Ang1) inhibited the early stages of VEGF- and Ang2-mediated angiogenesis if present simultaneously with VEGF and Ang2, but promoted these behaviors if added subsequently to the pro-angiogenesis factors. VEGF and Ang2 were also found to additively enhance microvessel density in a subcutaneous model of blood vessel formation, while simultaneously administered PDGF/Ang1 inhibited microvessel formation. However, a temporally controlled scaffold that released PDGF and Ang1 at a delay relative to VEGF/Ang2 promoted both vessel maturation and vascular remodeling without inhibiting sprouting angiogenesis. Our results demonstrate the importance of temporal control over signaling in promoting vascular growth, vessel maturation and vascular remodeling. Delivering multiple growth factors in combination and sequence could aid in creating tissue engineered constructs and therapies aimed at promoting healing after acute wounds and in chronic conditions such as diabetic ulcers and peripheral artery disease.     

从PBMCs及CD133免疫磁珠分选获得内皮祖细胞的体外研究

目的：比较从外周血单个核细胞(PBMCs)和CD133免疫磁珠分选体外培养人外周血内皮祖细胞(EPCs)的表型、分化、扩增、功能特点,为EPCs细胞治疗提供临床参考。方法： 健康成年人外周血,经Ficoll密度梯度离心法得PBMCs,经直接接种法或免疫磁珠分选CD133阳性细胞后置于M199培养基中培养,于第7、14 d比较2组细胞表型变化及细胞因子分泌、扩增、体外血管形成及趋化能力。结果：相同血量标本PBMCs组早期集落数高于CD133+组(P<0.01),随着培养时间的推移,流式细胞检测2组细胞均显示造血干细胞标志的表达下调和内皮细胞标志表达上升,但CD133+组内皮标志CD144表达率低于PBMCs组(P<0.01),ELISA法检测到在PBMCs组早期EPCs分泌VEGF的水平高于CD133+组(P<0.01),MTT法显示PBMCs组有较强的增殖能力,基质胶及Transwell实验表明PBMCs组细胞参与血管形成的能力较强。结论： CD133+来源的EPCs分化、分泌、增殖及血管形成能力相对较低,推断PBMCs中CD133-细胞可能在形成功能性的EPCs中发挥更重要的作用。     

Expression of leukocyte-endothelial cell adhesion molecules on monocyte adhesion to human endothelial cells on plasma treated PET and PTFE in vitro

We used a coculture model to evaluate the inflammatory potential of ammonia gas plasma modified PET and PTFE by flow cytometry and immunohistochemistry. In these studies, human endothelial cells from umbilical cord (HUVEC) and promonocytic U937 cells were used. HUVECs grown on polystyrene tissue culture coverslips and HUVECs stimulated with tumour necrosis factor (TNF-) were used as controls. U937 adhesion to endothelium on each surface was evaluated at day 1 and day 7. To further investigate the role of leukocyte–endothelial cell adhesion molecules (CAMs) in cell-to-cell interaction on material surfaces, the expression of the leukocyte–endothelial CAMs: ICAM-1, VCAM-1, PECAM-1, and E-selectin on HUVECs were evaluated after U937 cell adhesion. The results demonstrated that plasma treated PET (T-PET) and treated PTFE (T-PTFE) did not increase U937 cell adhesion compared to the negative control. Maximal adhesion of U937 cells to HUVEC was observed on TNF- stimulated endothelium with significant differences between day 1 and day 7, which is consistent with our prior observation that T-PET and T-PTFE did not cause HUVECs to increase the expression of adhesion molecules. After U937 cell adhesion, the expression of ICAM-1 and VCAM-1 of HUVECs were not different on T-PET and T-PTFE compared with the negative control. However, the expression of E-selectin was reduced on day 1, but not on day 7. The effects of plasma treated PET and PTFE on HUVEC adhesion and proliferation were also studied. On day 1 there were slight increases in the growth of HUVECs on both of T-PET and T-PTFE but this was not statistically significant. On day 7, the cell number increased significantly on the surfaces compared to the negative control. The results demonstrate that the plasma treatment of PET and PTFE with ammonia improves the adhesion and growth of endothelial cells and these surfaces do not exhibit a direct inflammatory effect in terms of monocyte adhesion and expression of leukocyte–endothelial CAMs. The monocyte adhesion to endothelial cells on surfaces can be used as a tool for the evaluation of material surface modification and further to study the mechanisms of cell-to-cell interactions in response to surfaces.     

细胞间黏附分子-1与血管生成的研究进展

实体肿瘤的生长、浸润和转移均依赖于血管生成这一进程,而血管生成是在一系列生长因子调控下实现的。有资料显示,细胞间黏附分子-1与血管生成有关,它通过与内皮细胞表面上的特异性受体结合而发挥其生物学活性,在某些疾病过程中的血管生成及发生发展过程中起着重要作用。现就细胞间黏附分子-1的生物学特性及与血管生成的关系研究进展概述如下。     

Morphological characteristics of cultured olfactory bulb cells

Cultured olfactory bulb cells from embryonic mice had ultrastructural characteristics similar to those of many cell types in the intact adult mouse olfactory bulb. Identified cultured cells included mitral/tufted cells, granule cells, short-axon cells, and fibrous and protoplasmic astrocytes. Cultured neurons were found as individual cells, clusters or aggregates. Clusters consisted of a loose array of neurons that appeared to be densely interconnected by neuntes. However, few neurites or fascicles emanated from clusters to adjoining areas. Aggregates consisted of many small, usually rounded, neurons piled on top of one larger neuron, or on more than one, with typically many neuntes and fascicles projecting to adjacent aggregates, clusters or individual neurons. Neurites of cultured olfactory bulb cells were well developed, and some were several millimeters long. Synapses were very prominent in these cultures, especially in aggregates, clusters, and fascicles. Electron-lucent, dense-core, and coated vesicles were present. Polarity, shape, and length of the long axis (size) of 815 cultured neurons, identified by positive anti-microtubule-associated protein 2 staining, were documented. Cultured neurons varied in size from 9 to 27 μm, with an average size of 16 μm. Elliptical bipolar (35%), triangular multipolar (21%), and round unipolar (15%) were the most common polarity/shape combinations found in culture. Multipolar, triangular, triangular multipolar, and elliptical bipolar cells increased in size with increasing age of culture. The relative proportions of triangular, multipolar, elliptical multipolar, and triangular multipolar cells decreased, whereas the relative proportions of round, unipolar, and round unipolar cells increased with increasing age of culture. These changes in population subtypes and cell size may indicate continued differentiation and maturation of cultured neurons.     

白藜芦醇对于外周血内皮祖细胞体外增殖分化的影响

目的:观察白藜芦醇对内皮祖细胞体外扩增分化过程中内皮系基因的表达及粘附、迁移功能的影响。方法:密度梯度离心法获得人外周血单个核细胞,在向内皮祖细胞诱导分化的同时,加入不同浓度的白藜芦醇(1、5、15、60μM)干预7天,随后采用流式细胞术观察CD34、CD31、KDR基因的表达;用RT-PCR和ELISA方法检测干预后内皮型一氧化氮合成酶的表达;同时测定干预后细胞的粘附和迁移功能。结果:低浓度白藜芦醇(5μM)不同程度促进内皮祖细胞增殖分化中CD34、CD31、KDR的表达,上调eNOS的mRNA和蛋白水平表达,并促进内皮祖细胞粘附和迁移功能;而高浓度白藜芦醇(60μM)则不同程度下调CD34、CD31、KDR、eNOS的表达和抑制粘附、迁移功能。结论:低浓度白藜芦醇可促进外周血内皮祖细胞内皮系基因的表达,并且促进其粘附和迁移,而高浓度则产生相反的作用。     

尿酸对外周血内皮祖细胞数量与功能的影响

目的：观察尿酸对外周血内皮祖细胞（endothelial progenitor cells,EPCs）数量与功能的影响。方法:采用Ficoll密度梯度离心法从外周血获得单个核细胞,将其接种于包被人纤维连接蛋白的培养板上,培养14 d后收集贴壁细胞,加入不同浓度(50 mg/L,150 mg/L,300 mg/L)尿酸分别培养24 h、48 h、72 h。激光共聚焦显微镜鉴定,FITC-UEA-I 和Dil-acLDL 双染色阳性细胞为正在分化的EPCs,并在倒置荧光显微镜下计数。然后分别采用MTT 比色法、黏附能力测定实验来观察EPCs 的增殖能力、黏附能力。结果:尿酸呈量效和时效地增加EPCs 数量，300 mg/L尿酸作用48 h后EPCs数量明显多于对照组（32.5±3.6 vs 78.2±4.1，P< 0.01），并增加EPCs增殖能力（0.179±0.005） vs （0.322±0.011）, P< 0.01，和黏附能力（20.5±3.7） vs （67.8±4.9）, P< 0.01。结论:尿酸可增加EPCs 数量并改善EPCs的增殖及黏附能力。