Correlating fibronectin adsorption with endothelial cell adhesion and signaling on polymer substrates

Fibronectin (Fn) adsorption was studied on different commercial polymer surface chemistries, including tissue culture polystyrene (TCPS), bacteriologic polystyrene (BPS), fluoropolymer Teflon AF, and poly-L-lactide (PLLA). Antibody probes detected the availability of Fn's cell binding domain on adsorbed Fn in the competitive presence and absence of bovine serum albumin (BSA). Domain availability was highest for Fn adsorbed on TCPS, especially in the presence of either serum albumin or dilute serum. Attachment and growth efficiencies for human umbilical venous endothelial cells (HUVECs) cultured on surfaces preadsorbed with Fn in serum and serum-free media correlated with antibody cell-binding domain availability: TCPS > BPS, Teflon AF > PLLA. Intracellular signaling from the GTPase, RhoA, was highest (RhoA:RhoGDI inhibitor ratio) in cells cultured on the Teflon AF surfaces, indicating that despite lower attached cell numbers on Teflon AF compared to TCPS, cell signaling remained activated after 24 h of growth. Up-regulated cellular Fn mRNA messages, assessed using RT-PCR techniques, supported HUVECs' producing the endogenous extracellular matrix (ECM) protein Fn in order to attach and survive on the suboptimal Teflon AF culture surfaces.     

十二烷基化壳聚糖基因支架血管内基因转运的实验研究

目的评估新型十二烷基化壳聚糖质粒DNA支架向局部动脉血管内转运基因的可行性及效果。方法十二烷基化壳聚糖同质粒DNA按文献制备纳米粒并进行表征。将纳米粒涂布于金属支架表面同时进行电镜观察。分别将十二烷基化壳聚糖质粒DNA支架进行细胞转染(pEGFP-C1)和兔颈动脉在体转染实验(pGL3-control),观察细胞转染效果。结果十二烷基化壳聚糖质粒DNA纳米粒为球形,稳定,平均粒径126nm,Zeta电位( 28±3)mV。该纳米粒涂布于支架表面,电镜下呈片状不规则粒子。细胞转染实验显示,支架表面细胞及支架邻近细胞大量转染,远离支架细胞未见转染。动物实验结果显示,支架植入部位荧光素酶高表达,同时有2只动物肝脏标本有微量表达,其他部位未见表达。结论十二烷基化壳聚糖质粒DNA支架是一种新型有效的血管内基因转运体系,可能对诸如再狭窄等心血管疾病有良好的应用前景,同时,这一基因运载思路可以广泛应用于各种质粒DNA的转运。     

Interactions of dendritic cells with fibronectin and endothelial cells.

We studied the phenotypic characteristics of spontaneously migrated skin dendritic cells (sDC) and monocyte-derived dendritic cells (moDC), generated under different culture conditions, and their interactions with fibronectin (FN) and endothelial cells. Monocyte-derived dendritic cells were obtained after culturing monocytes with granulocyte-macrophage colony-stimulating factor (GM-CSF) (800 U/ml) and interleukin-4 (IL-4) (500 U/ml) with either 10% fetal bovine serum (FBS) or 10% allogeneic human serum (HS). Regardless of the type of serum used, the majority of moDC expressed human leucocyte antigen-DR (HLA-DR) and CD86. On day 5 of incubation, 20-67% of moDC cultured in the presence of HS (HS-moDC) expressed CD1a, b and c versus 94-97% when cultured in the presence of FBS (FBS-moDC). DC showed a differential gradient of adhesion to FN: FBS-moDC>HS-moDC>sDC approximately monocytes. Both FBS-moDC and HS-moDC were strongly positive for CD49e (alpha5-integrin) and CD29 (beta1-integrin) but negative for CD49d (alpha4-integrin). A monoclonal antibody (mAb) against CD49e blocked the adhesion of both types of moDC to FN. Although both FBS-moDC and HS-moDC attached to endothelium (a 76% and 63% increase, respectively), only HS-moDC were able to migrate through non-activated endothelium. Overall, these results suggest that spontaneously migrated sDC are less adherent to FN than moDC, that HS and FBS induce differences in CD1 expression, that HS-moDC are less adhesive to FN and endothelial cells but more motile than FBS-moDC, and that alpha5beta1-integrin is the molecule involved in moDC adhesion to FN.     

Effect of plasma fibronectin on the adhesive properties of human peripheral lymphocytes

The adhesive properties of human peripheral lymphocytes (HPL) towards immunobilized substrate in vitro have been investigated. The role of plasma fibronectin was studied in two different systems: a) immobilized fibronectin as a model of cell adhesion in the tissues; b) soluble fibronectin as a model for cell adhesion in blood circulation. Collagen (Type I) or collagen-fibronectin-coated surfaces were used as substrate for lymphocyte adhesion in system b. A very small amount of HPL (5.1 +/- 1.2%) was attached to the collagen surface. The adhesion to fibronectin or to collagen-fibronectin surfaces, however, was substantially higher, reaching a value of 34.1 +/- 3.8% and 32.7 +/- 5.8%, respectively. The addition of soluble fibronectin to system a leads to a competitive inhibition of adhesion. On the contrary, in the presence of soluble fibronectin, the augmentation of the lymphocyte adhesion to collagen was found to be dose-dependent and nearly 4 times higher as compared to the control level. This effect was found to be maximal at fibronectin concentrations of 250-300 micrograms/ml. The data demonstrate the important role of plasma fibronectin in the adhesive interactions of lymphocytes.     

The influence of adhesive and invasive properties of Staphylococcus aureus defective in fibronectin-binding proteins on secretion of interleukin-6 by human endothelial cells

Fibronectin-binding proteins (FnBP) are surface adhesins of Staphylococcus aureus documented to be virulence attributes in, for example, endovascular infections. By using mutants of S. aureus defective in the FnBPA and B genes we have investigated whether these adhesins affect cytokine expression in human umbilical vein endothelial cells (HUVEC). S. aureus expressing FnBPA and B adhered to and were internalized into HUVEC to a greater extent compared to mutants defective in expression of FnBP. Production and release of IL-6 was higher from endothelial cells infected with the parent FnBP-expressing strain compared to the FnBP-defective mutants. These results indicate that adhesion to and invasion of S. aureus into endothelial cells are important regulators of cytokine expression.     

Development of a novel endothelial cell-seeded endovascular stent for intracranial aneurysm therapy

The metallic stent has been widely used in endovascular treatment of intracranial aneurysms and arterial stenosis. Endothelialization at the neck of the aneurysm or stenotic lesion after stent deployment plays a pivotal role in preventing aneurysm recurrence, as well as local thrombus formation and restenosis. To deliver autologous endothelial cells and to promote the endothelialization on the luminal wall of the parent artery, we established an endothelial cell-seeded intracranial stent device. Endothelial cells were isolated from canine jugular vein and identified by FACS assay and immunohistochemistry. We demonstrated that the seeded endothelial cells formed a confluent endothelial layer on the stent's surface. After being brushed with 100 dyne/cm(2) of shear stress, we found that this endothelial layer remained intact for at least 48 h on the heparinized polymer coated stent, rather than the poly-lactic-acid coated stent (p < 0.05). The results suggest that an autologous endothelial cell-seeded stent may be a feasible and optimal tool for endothelial delivery during stenting and may overcome some limitations of the traditional bare stent in the treatment of intracranial aneurysms and arterial stenosis.     

The effect of cyclic mechanical strain on activation of dendritic cells cultured on adhesive substrates

Dendritic cells (DCs), key regulators of tolerance and immunity, have been found to reside in mechanically active tissues such as the interior layers of the arterial wall, which experience cyclic radial wall strain due to pulsatile blood flow. Although experimentally difficult to determine in vivo, it is reasonable to postulate DCs experience the mechanical forces in such mechanically active tissues. However, it is currently unknown how DCs respond to cyclic mechanical strain. In order to explore the hypothesis that DCs are responsive to mechanical strain, DCs were cultured in vitro on pre-adsorbed adhesive proteins (e.g., laminin, collagen, fibrinogen) and 1 Hz cyclic strain was applied for various durations and strain magnitudes. It was determined that a strain magnitude of 10% and 24 h duration adversely affected DC viability compared to no-strain controls, but culture on certain adhesive substrates provided modest protection of viability under this harsh strain regime. In contrast, application of 1 h of 1 Hz cyclic 3% strain did not affect DC viability and this strain regime was used for the remaining experiments for quantifying DC activation and T-cell priming capability. Application of 3% strain increased expression of stimulatory (MHC-II) and costimulatory molecules (CD86, CD40), and this effect was generally increased by culture on pre-coated adhesive substrates. Interestingly, the cytokine secretion profile of DCs was not significantly affected by strain. Lastly, strained DCs demonstrated increased stimulation of allogeneic T-cell proliferation, in a manner that was independent of the adhesive substrate. These observations indicate generation of a DC consistent with what has been described as a semi-mature phenotype. This work begins elucidating a potential role for DCs in tissue environments exposed to cyclic mechanical forces.     

Comparison of endothelial cells grown on different stent materials

We compared the behavior of endothelial cells grown on stent materials. Human umbilical vein endothelial cells (HUVEC) were seeded (200 or 800 cells/mm(2)) onto different metallic sheets, including 316 stainless steel (low carbon; 316LSS), nitinol, and 316LSS coated with TiN or TiO(2). Cells seeded onto tissue culture-treated polystyrene dish coated with gelatin were used as controls. Forty-eight hours later, the cells were examined by Western blotting, immunofluorescence microscopy, and scanning electron microscopy (SEM). The results showed that for either seeding values, the levels of cellularity on TiN and TiO(2) are comparable or higher, and those on 316LSS and nitinol are lower compared to the controls (p < 0.05). SEM demonstrated that cells are well-attached on the metallic surface with various amount of cellular processes. In metals seeded with 800 cells/mm(2), Western blotting showed that the overlying cells expressed less amounts of endothelial nitric oxide synthase (eNOS), Von Willebrand factor (VWF), and connexin43 protein compared to the controls (p < 0.05). Immunofluorescence microscopy confirmed the results of immunoblotting. In conclusion, stent materials affect HUVEC's growth and protein expression profile. Down-regulation of eNOS, VWF, and connexin43 gap junctions is a common phenomenon in the cells growing on the examined metallic materials, suggesting the existence of endothelial dysfunction in the arterial segments containing the stents made of such materials.     

SSeCKS对牛肺动脉内皮细胞黏附和迁移的影响

目的： 研究SSeCKS在细胞外基质成份诱导内皮细胞黏附和迁移中的作用。方法： 用纤黏连蛋白(FN)诱导内皮细胞，以Western blotting分析SSeCKS表达量的变化；用Ro31-8220、calphostin C（蛋白激酶C抑制剂）预处理内皮细胞，观察对细胞黏附和迁移的影响，以共聚焦显微镜观察其对SSeCKS、F-actin和vinculin在细胞定位的影响。结果： FN能够显著诱导内皮细胞黏附和迁移，SSeCKS的表达量也随之增加，具有时间和剂量依赖性；经Ro31-8220、calphostin C处理细胞后，SSeCKS表达量明显减少，细胞黏附率和迁移细胞数也较处理前显著减少，SSeCKS由细胞质散在分布向核周聚集，且SSeCKS与F-actin、vinculin在细胞边缘共定位比处理前减少。结论： SSeCKS参与细胞外基质诱导内皮细胞黏附和迁移的过程，由其介导的PKC信号转导促进了这一过程，蛋白激酶C抑制剂可有效抑制此过程。     

Cultivation of endothelial cells on adhesive protein-free synthetic polymer gels

Various hydrogels without modification by any cell adhesive proteins have been investigated as cell scaffolds. The present study shows that bovine fetal aorta endothelial cells can adhere, spread, proliferate, and reach confluence on poly(acrylic acid), poly(sodium p-styrene sulfonate), and poly(2-acrylamido-2- methyl-1-propanesulfonic sodium) gels, whereas cells reach subconfluence on poly(vinyl alcohol) and poly(methacrylic acid) gels. The proliferation behavior was sensitive to both hydrogel charge density and crosslinker concentration. The relationship between cell proliferation and zeta potential of gels was discussed. It was found that hydrogels with a negative zeta potential higher than about 20 mV facilitates cell proliferation.     

肾小管上皮细胞与基底膜的粘附力学特性

目的：研究模拟缺血、缺氧、缺血缺氧三种条件下肾小管上皮细胞与基底膜的粘附力学特性。材料与方法：利用微管吸吮技术测定肾小管上皮细胞与人工基底膜的粘附力。结果：模拟缺血、缺氧、缺血缺氧三种模型中肾小管上皮细胞与基底膜的粘附力均较正常情况明显降低。结论：不同模拟损伤因素对粘附力的影响不同，其中缺血缺氧组粘附力最小，单纯缺血组的影响较小。这些结果提示，在一定条件下，氧缺乏比缺血情况更能影响肾小管上皮细胞与     

Deposition of endothelial fibronectin on polymeric surfaces

Cellular fibronectin is deposited on tissue culture polystyrene during the adhesion and spreading of cultured human endothelial cells (HEC). Following the seeding of HEC upon this polymer, larger amounts of fibronectin are deposited as both cell density and incubation time increase. Our results indicate that the ability to deposit cellular fibronectin onto a polymeric surface is a condition for the spreading and proliferation of HEC.     

The effect of vessel material properties and pulsatile wall motion on the fixation of a proximal stent of an endovascular graft

Migration is a serious failure mechanism associated with endovascular abdominal aortic aneurysm (AAA) repair (EVAR). The effect of vessel material properties and pulsatile wall motion on stent fixation has not been previously investigated. A proximal stent from a commercially available stent graft was implanted into the proximal neck of silicone rubber abdominal aortic aneurysm models of varying proximal neck stiffness (β=25.39 and 20.44). The stent was then dislodged by placing distal force on the stent struts. The peak force to completely dislodge the stent was measured using a loadcell. Dislodgment was performed at ambient pressure with no flow (NF) and during pulsatile flow (PF) at pressures of 120/80 mmHg and 140/100 mmHg to determine if pulsatile wall motions affected the dislodgement force. An imaging analysis was performed at ambient pressure and at pressures of 120 mmHg and 140 mmHg to investigate diameter changes on the model due to the radial force of the stent and internal pressurisation. Stent displacement forces were ~50% higher in the stiffer model (7.16-8.4 N) than in the more compliant model (3.67-4.21 N). The mean displacement force was significantly reduced by 10.95-12.83% from the case of NF to the case of PF at 120/80 mmHg. A further increase in pressure to 140/120 mmHg had no significant effect on the displacement force. The imaging analysis showed that the diameter in the region of the stent was 0.37 mm greater in the less stiff model at all the pressures which could reduce the fixation of the stent. The results suggest that the fixation of passively fixated aortic stents could be comprised in more compliant walls and that pulsatile motions of the wall can reduce the maximum stent fixation.     

CD226分子参与内皮细胞黏附功能的形态学证据

目的：提供CD226分子参与内皮细胞黏附功能的形态学证据。方法：体外培养人脐静脉内皮细胞，加入融合蛋白CD226—Ig，采用倒置显微镜和扫描电子显微镜观测CD226分子在活化内皮细胞和活化PBMC黏附过程中的作用。结果：融合蛋白CD226／Ig可显著降低活化内皮细胞与活化PBMC间的黏附。结论：CD226分子是活化内皮细胞表面一种新的黏附分子，可能具有重要的生理和病理功能。     

管腔内支架人工血管治疗主动脉夹层

目的 探讨应用腔内支架人工血管植入治疗DeBakeyⅢ型主动脉夹层的适应症、操作技巧及并发症防治。方法 对18例DeBakeyⅢ型主动脉夹层在全麻下行腔内支架人工血管植入术，经左上肢置入造影导管造影，经股动脉导入支架人工血管植于夹层破口处封闭破裂口。结果18例共植入支架人工血管20只，2例发生内漏，均经加放一只支架人工血管封堵成功。操作成功率100％，无手术死亡。近期、中期效果良好。结论 支架人工血管植入术治疗DeBakeyⅢ型主动脉夹层具有创伤小，恢复快的特点，尤其适用于病人体质差，有合并症及外科手术禁忌的病人，近期效果良好．远期效果有待观察。     

Detection by ELISA of laminin released by endothelial cells from a collagen coated layer of Matrigel

Summary We have developed an in vitro model system quantitating the degradation of extracellular matrix by endothelial cells. Collagen is layered over a reconstituted basement membrane matrix (Matrigel) and the release of laminin contained in the Matrigel into the media by endothelial cells is quantitated by ELISA. The cell-specific release of laminin is consistent and reproducible. Incubating an equivalent number of dead endothelial cells in the same manner results in no release of laminin relative to media controls. The cell-specific laminin release is abolished by removing the exogenous growth factor component of the media and plating the cells in media plus 10% serum. Cells plated onto wells coated with collagen alone do not release laminin into the media, indicating that no de novo synthesis and release of laminin occurs during the time frame of the experiment, and that some factor or combination of factors present in the media supplement is essential for the endothelial cells to release laminin degrading enzymes.     

Expression of adhesion molecules and cytokines in vitro by endothelial cells seeded on various polymer surfaces coated with titaniumcarboxonitride

Although endothelial cell (EC) seeding improves the patency of vascular prostheses, the detachment of adherent ECs after the restoration of circulation remains one of the major obstacles. Polymer surfaces for endothelialization can be optimized. In this study, polyethylene terephthalate (PET), polypropylene (PP), polytetrafluoroethylene (PTFE), polyurethane (PUR), and silicone were coated with a titaniumcarboxonitride (Ti(C,N,O)) layer by a plasma-assisted chemical vapor deposition process to verify the effect of titanium onto human saphenous vein ECs. Almost confluent EC monolayers were evaluated for 1) proliferation activity and 2) expression of adhesion molecules using cellular enzyme-linked immunosorbent assay and release of cytokines. The results showed that all titanium-coated polymers and uncoated PET have no toxic effect on human saphenous vein ECs excepting uncoated PTFE, PP, and silicone. Moreover, growing ECs showed an insignificant decrease in cytokine production and an unessential change in basal expression of adhesion molecules. Tumor necrosis factor-alpha-induced response depends on polymer surface: for example, intercellular adhesion molecule-1 expression decreased. E-selectin expression was unchanged for culturing ECs on coated PET, PP, and PTFE and reduced for polyurethane and silicone. Vascular cell adhesion molecule-1 expression was unchanged for coated PUR and silicone and reduced for PET, PP, and PTFE. In summary, titanium-coating layers promote adhesion of human ECs on polymer vascular grafts with no proinflammatory reaction of ECs.     

内皮细胞中一种新的可溶型黏附分子sCD226的表达调变规律

目的：探讨人内皮细胞中一种新的可溶型黏附分子sCD226的表达调变规律。方法：用ELISA方法，检测不同浓度的LPS刺激人脐静脉内皮细胞后不同时间点培养上清中sCD226含量的变化；用Greiss法检测上述细胞培养上清中N0含量的变化，并分析两者的线性相关性。加入抗TNF-α中和抗体和iNOS抑制剂后，观察其对LPS诱导sCD226生成的影响。结果：①随着LPS刺激剂量的增加和刺激时间的延长，HUVEC培养上清中sCD226的含量明显增加，100mg／L LPSS刺激3d后sCD226的含量为188．5μg／L。②LPS刺激剂量为100mg／L时，HUVEC培养上清中sCD226和N0的含量呈显著的正相关。③抗TNF—α中和抗体和iNOS抑制剂可显著抑制LPS活化的HUVEC产生sCD226。结论：LPS可诱导HUVEC产生sCD226分子，且sCD226的生成与N0和TNF-α的产生密切相关。