Endothelial Cell Seeding of Small Diameter Vascular Grafts

Abstract: This study examines, under flow conditions, the adhesion of endothelial cells to 3 mm diameter fi-bronectin (Fn)-coated expanded polytetrafluoroethylene (PTFE) vascular grafts. Cultured ovine carotid artery endothelial cells were labelled with 35S-methionine. The grafts were seeded with endothelial cells (1.5 times 106/ml) by rolling for 1 h at 37C and then either cultured to confluence for 48 h or flow tested immediately. Cell attachment to grafts (n = 5) was evaluated in an in vitro flow circuit, using flows of up to 330 ml/min. Ex vivo studies (n = 5 grafts) were conducted without anticoagulant using autologous cells in a sheep model. Grafts were inserted into an externalized carotid-jugular shunt and exposed to blood flows of ˜150 ml/min for 3 h. One hour seeded and 48 h cultured grafts demonstrated <95% cell retention following in vitro flow studies. Ex vivo studies of 48 h cultured grafts gave endothelial cell retention of 81% with no sign of thrombogenicity. Furthermore, a preliminary 24 h ex vivo study has shown <95% retention. This study demonstrates the firm attachment of seeded endothelial cells to Fn-coated PTFE grafts in the sheep model.     

基因工程内皮细胞种植人工血管的研究进展

自体和非自体的生物血管不能满足临床血管外科的需要。使用理化方法或单纯种植内皮细胞的方法处理人工血管因腔内血栓形成而效果小理想。为了增加人工血管的抗血栓活性，利用基因工程的方法对内皮细胞进行改造，可使其获得稳定表达抗凝血因子的能力。用这种基因工程改造的内皮细胞种植于人工血管，有可能获得较理想的效果。而最终要获得理想的血管移植物，尚有许多需要解决的问题。     

Galectin-3对外周血内皮祖细胞源性血管内皮细胞增殖能力的影响

目的观察重组人半乳糖凝集素-3(Galectin-3)对外周血内皮祖细胞源性血管内皮细胞增殖能力的影响。方法提取人外周血内皮祖细胞,将其定向诱导为成熟血管内皮细胞,加入不同终浓度的Galectin-3进行培养,观察不同浓度Galectin-3对外周血内皮祖细胞源性内皮细胞增殖能力的影响。结果 0.1、1.0、2.5、5.0和10.0μg/ml浓度组外周血内皮祖细胞源性内皮细胞的增殖能力均高于0μg/ml组,其中0.1,1.0及2.5μg/ml浓度组与0μg/ml组比较差异无统计学意义(P>0.05),而5.0及10.0μg/ml组细胞增殖能力明显高于0、0.1、1.0及2.5μg/ml组,差异有统计学意义(P<0.05),10.0μg/ml浓度组细胞增殖能力又明显高于5.0μg/ml浓度组,差异有统计学意义(P<0.05)。结论 Galectin-3可促进体外培养的外周血内皮祖细胞源性血管内皮细胞的增殖能力。     

Endothelial Cell Seeding on Different Polyurethanes

Six polyurethanes (PUs) were tested with respect to their cytocompatibility. Initial adhesion, initial spreading, and the proliferation of endothelial cells were investigated. All smooth PUs showed similar initial adhesion. Initial spreading was faster on rough PUs. Collagen coating resulted in faster initial adhesion but not better proliferation of endothelial cells.     

Vascular Prosthesis Standards: A Manufacturer''s Viewpoint

A review of the state of current commercial vascular grafts and the types of tests proposed by the Association for the Advancement of Medical Instrumentation Vascular Prosthesis Draft Standard is presented. The philosophy of the standard and the types of products covered are discussed. Several areas requiring additional development are noted. The status and future of the standard are now up to the general user community.     

Adhesion and Stability of Fibronectin on PTFE Before and After Seeding with Normal and Synchronized Endothelial Cells: In Vitro Study

Abstract: The possible influence of the cell cycle on the efficacy of endothelial cell (EC) seeding onto the surface of polytetrafluoroethylene (PTFE) prostheses was studied. Likewise the ideal fibronectin concentration and optimal incubation time to guarantee the binding of this protein to the prosthetic surface have been calculated. Synchronized ECs, previously labeled with ³H-thymi-dine, were used, and the loss of radioactivity was determined at several times throughout the study. The results showed a progressive loss of cells on the prosthetic surface similar to that occurring with the seeding of un-synchronized ECs. The optimal concentration of fibronectin was 20 μg/ml, and the optimal incubation time was 1 h.     

Growth Regulation of Endothelial Cells by Tissue Fragments: Analysis on Mechanism of Rapid Endothelialization of Tissue-Seeded Vascular Prosthesis in a Three-Dimensional Culture System

Abstract: Rapid endothelialization of the inner surface was reported in an autologous tissue-seeded vascular prosthesis. We applied a three-dimensional in vitro culture system to elucidate the precise mechanism of rapid endothelial coverage of a tissue-seeded vascular prosthesis. Human venous, omental, adipose and striated muscle tissue fragments were harvested from surgical specimens. They were embedded in collagen gel, and 2.0 times 10⁵ bovine aortic endothelial cells (BAECs) were seeded on the gel. The number of BAECs was counted on Days 2 and 7. Growth rate of BAECs was facilitated on the collagen gel with omental and striated muscle tissue fragments (p < 0.05). Factor VIII-negative spindle cells migrated around tissue fragments, especially around the omental and striated muscle tissue fragments. Rapid endothelialization of a tissue-seeded vascular prosthesis may result from facilitation of EC proliferation by viable tissue fragments and migrated cells. These results confirm tissue fragments regulate EC growth, and are useful as bioengineering tools     

Fibronectin Coating of Expanded Polytetrafluoroethylene (ePTFE) Grafts and Its Role in Endothelial Seeding

Although fibronectin's role as a matrix to improve endothelial seeding has been demonstrated by other workers, the optimum concentration for use has never been described. Attachment of fibronectin to expanded polytetrafluoroethylene (ePTFE) was measured, using 125I-radiolabeled protein, at different concentrations and for different time periods. The absolute amount of fibronectin bound to the graft increased with the concentrations used in coating (p less than 0.001) and also with time (p less than 0.01); e.g., at 50 micrograms/ml, 90 min of incubation produced a molecular attachment of 4.0 x 10(11)/cm2 of graft. However, its percentage attachment decreased with a rise in concentration (p less than 0.001). After an initial loss of 22% in 30 min, the fibronectin-graft bond was found to be stable when exposed to a shear stress produced by flow at 200 ml/min. No significant difference in the cell adherence could be found in grafts coated with fibronectin concentrations of 50, 150, and 250 micrograms/ml, although it was significantly less at 10 and 25 micrograms/ml (p less than 0.05).     

In vivo patency of endothelial cell-lined expanded polytetrafluoroethylene prostheses in an ovine model

The performance of small-diameter vascular prostheses may be improved by implantation of grafts lined with endothelial cells. Expanded polytetrafluoroethylene (ePTFE) prostheses (4 mm x 40 mm) were coated with fibronectin (20 micrograms/ml), seeded with endothelial cells, and cultured for 48 h to produce a confluent, autologous endothelial cell lining. They were implanted as carotid interposition grafts in sheep. Seeded ePTFE grafts were compared with nonseeded ePTFE grafts and autologous carotid artery grafts. No anticoagulant or antiplatelet therapy was administered, making this a stringent test model for the thromboresistance of a small-diameter prosthesis. After 13 weeks the patencies of seeded, nonseeded, and autologous artery grafts were 16% (1/6), 0% (0/6), and 100% (6/6), respectively. The one seeded graft that was patent was fully lined with endothelial cells and showed no stenosis. The remaining five seeded grafts were occluded by fibrous tissue and displayed substantial spindle cell hyperplasia. There was no apparent difference between the autologous artery grafts and normal arterial tissue, and the anastomoses showed no stenosis. The ovine model provides a conservative test of prosthesis survival and may be useful for study of graft failure.     

Endothelial Cell Retention on a Viscoelastic Nanocomposite Vascular Conduit Is Improved by Exposure to Shear Stress Preconditioning Prior to Physiological Flow

In this study, endothelial cell (EC)‐seeded nanocomposite grafts were preconditioned with 1–2 dynes/cm² in vitro to establish whether low shear stress resulted in improved cell adherence prior to physiological shear stress (15 dynes/cm²). Alamar blue cell viability was assessed. Polymerase chain reaction was conducted for glyceraldehyde‐3‐phosphate dehydrogenase, transforming growth factor beta‐1 (TGFβ‐1), vascular endothelial growth factor receptor‐1 (VEGFR‐1), platelet EC adhesion molecule‐1, and vascular endothelial growth factor receptor‐2 (VEGFR‐2). The Alamar blue results demonstrated improved cellular retention following preconditioning (P < 0.001). VEGFR‐2 and TGFβ‐1 expression was up‐regulated, and VEGFR‐1 down‐regulated following preconditioning. This investigation confirms previous findings regarding the potential benefits of preconditioning, and demonstrates that these benefits can be applied to ECs seeded on the nanocomposite employed. It also demonstrates further the suitability and potential of nanocomposite for future use in tissue‐engineered cardiovascular devices.     

Effectiveness of Endothelial Cell Seeding on Patency of Damaged Vascular Surfaces in a Canine Model

The effects of endothelial cell seeding, which is assumed to be an effective technique to improve patency rates of denuded vascular surfaces, were investigated in an experimental model. In this study, after anesthetic induction, jugular veins of 16 dogs were harvested bilaterally. Endothelial cells were extracted enzymatically by collagenase from these veins and were passaged into a culture medium until they grew to a reasonable number. After 3 weeks, dogs were anesthetized again in a similar fashion and bilateral femoral veins were exposed and experimental intimal denudation was performed. Subsequently, one femoral artery was injected with cell solution and the other with saline solution as a control. Two weeks after the injections, arteriographic studies of femoral arteries were performed and arterial specimens were taken for histological evaluation. Our results suggest that endothelial seeding might improve the patency rate in elective but urgent cases in which endarterectomy, percutaneous transluminal angioplasty, or similar vascular procedures are considered.     

血管内皮生长因子165基因重组腺病毒载体的构建及转染内皮祖细胞的实验研究

目的：利用细菌内同源重组法构建含血管内皮生长因子（VEGF）165基因的重组腺病毒，并观察其在内皮祖细胞（EPCs）中的表达，为进一步研究其在慢性肾脏病中的作用奠定基础。方法：用限制性内切酶XbaⅠ＋HindⅢ从质粒载体中切出VEGFl65基因片段，与KpnⅠ＋HindⅢ双酶切的pAdTrack-CMV形成转移质粒pAdTrack-CMV-VEGFl65，PmeI酶切线性化后与腺病毒基因组质粒pAdEasy-1共转化大肠杆菌BJ5183，由人胚肾293细胞包装成Ad-VEGFl65，PCR和westem blot法鉴定其表达。贴壁法体外培养获得大鼠骨髓来源的EPCs，Ad-VEGFl65基因体外转染EPCs，检测转染后目的基因的表达情况。结果：利用CSCI，法由pAdTrack-VEGFl65和pAdEasy-1共转化BJ5183感受态茵，可获得阳性重组体细菌克隆。PCR检测表明重组腺病毒已含有目的基因，病毒滴度1．4×10^10pfu／ml。Ad-VEGFl65转染培养第6天的EPCs后24h开始，培养上清中VEGF蛋白表达较对照组和转染前增加。结论：成功制备的重组体腺病毒Ad-VEGFl65转染体外培养的EPCs后，在体外能有效高表达目的基因产物，为今后VEGF在肾脏病中的深入研究奠定了基础。     

Cancer Cells Cause Vascular Endothelial Cell (vEC) Retraction via 12(S)HETE Secretion; The Possible Role of Cancer Cell Derived Microparticle

Background Cancer cell mediated vascular endothelial cell (vEC) retraction plays a pivotal role in cancer metastasis. The aim of this study is to clarify the biochemical character of vEC retraction factor derived from human breast cancer cell line, MCF-7. Methods and Results In order to estimate vEC retracting activity, transwell chamber assay system was employed. We first tested the effects of trypsin digestion as well as lipid extraction of culture medium (CM). Trypsin digestion of CM resulted in approximately 40% loss of vEC retracting activity and lipid extraction of CM by Brigh and Dyer methods recovered approximately 60% of vEC retracting activity, suggesting that approximately 60% of vEC retracting activity in MCF-7 derived CM is due to lipid. Although Nordihydroguaiaretic acid (NDGA), the specific lipoxygenase inhibitor, suppressed vEC retracting activity in CM, Acetyl salicylic acid (ASA), a specific cyclooxygenase inhibitor, did not affect the activity, suggesting that lipid exerting vEC retracting activity in CM belongs to lipoxygenase mediated arachidonate metabolites. Thin layer chromatography clearly demonstrated that Rf value of lipid vEC retracting factor in CM is identical to 12HETE. Authentic 12(S)HETE, but not 12(R)HETE, showed vEC retracting activity. After the ultracentrifugation of CM, most lipid vEC retracting activity was recovered from the pellet fraction, and flow cytometric analysis using specific antibody against 12(S)HETE clearly showed the association of 12(S)HETE with small particle in CM. Conclusion These findings suggested the principal involvement of 12(S)HETE in cancer cell derived microparticles in cancer cell mediated vEC retraction.     

Rabbit Model for Evaluation of a Long Small-Caliber Vascular Graft

We developed an experimental method for evaluating the properties and healing process of a long, small-caliber vascular graft in a small animal. Eight rabbits were used. A left thoracotomy was performed and the thoracic aorta was isolated. The aorta was clamped and excised in the middle without a temporary shunt. A 15-cm-long by 4.0-mm-ID segment of a vascular graft was implanted end to end. After the blood flow of the aorta was restarted, the long graft remained in a big loop shape in the thoracic cavity and showed no tension. The major advantage of this technique is that, although it is simple, we are still able to evaluate a long, small-caliber vascular graft in a small animal.     

小剂量脂多糖诱导血管内皮细胞TLR4的表达

目的:观察小剂量脂多糖(LPS)对人脐静脉内皮细胞(ECV304)TOLL样受体4(TLR4)表达的影响。方法:体外培养ECV304细胞,分别与LPS及LPS+TLR4抗体进行孵育。MTT法检测细胞的增殖活性,免疫组化染色法检测细胞表面TLR4的表达,实时荧光定量聚合酶链反应(RT-PCR)检测细胞核核内TLR4-mRNA及IL-8mRNA的表达。结果:LPS(10～50ng/mL)刺激ECV304细胞24h内,细胞增殖活性无明显变化(P>0.05);而以100ng/mL刺激24h后,细胞增殖活性明显降低(P<0.05),TLR4抗体对此无明显拮抗作用。LPS能明显上调ECV304表达TLR4、TLR4-mRNA及IL-8mRNA,其中10ng/mL的LPS在24h时、50ng/mLLPS在6～24h时,TLR4表达具有统计学意义(P<0.05);50ng/mL的LPS刺激ECV304细胞在4h及8h时,细胞核内TLR4mRNA表达均明显升高(P<0.05),而IL-8mRNA表达在8h时明显升高(P<0.05)。TLR4抗体对ECV304表达TLR4及TLR4-mRNA有拮抗作用(P<0.05),对IL-8mRNA表达无明显拮抗作用(P>0.05)。结论:小剂量LPS可诱导ECV304细胞表达TLR4并引起细胞活化,TLR4抗体可抑制TLR4的表达,但不能抑制细胞的活化。     

Endothelial progenitor cells for postnatal vasculogenesis

Bone marrow-derived endothelial progenitor cells (EPCs) are present in the systemic circulation, are augmented in response to certain cytokines and/or tissue ischemia, and are home to – as well as incorporate into – sites of neovascularization. On the basis of these aspects, EPCs have attractive potential therapeutic applications for cardiovascular ischemic diseases as a novel cell-based strategy, mainly via a vasculogenesis mechanism. This review provides an update of the biology of EPCs, as well as highlighting the potential use of these cells for therapeutic regeneration.     

Effect of Cilostazol on Endothelial Cell Denudation and Proliferation in Canine Vein Grafts

The purpose of the present study was to examine the effects of cilostazol on endothelial cell denudation and proliferation in vein grafts used as arterial substitutes. Unilateral aortoiliac bypass was performed using the lateral jugular vein in 20 mongrel dogs. The animals were divided into two groups according to whether or not cilostazol was given. The grafts were removed at intervals of 1 day and 50 days, and the luminal surface was assessed for endothelial cell coverage (%). The denudation of endothelial cells was less extensive in the cilostazol group than in the control group on postoperative day 1. There was significantly more proliferation of endothelial cells in the control group over the course of time than in the cilostazol group. In conclusion, cilostazol significantly prevented early endothelial cell denudation, although it did not appear to stimulate successive endothelial cell proliferation. Therefore, cilostazol may help preserve an intact intima, which would potentially result in the inhibition of intimal hyperplasia. Received: October 6, 2000 / Accepted: May 15, 2001     

Vascular Access Device for Treatment of Cancer Patients

Abstract: Vascular access devices that are completely implanted have been used for treatment of cancer patients. Vascular access devices are useful for transarterial infusion of anticancer drugs, intravenous hyperalimentation, and drainage of bile juice in obstructive jaundice. These systems have several advantages in the care of patients: they are sealed, they have no external tubes, and they may be useful for blood or biliary sample and intravenous hyperalimentation or chemotherapy. There are only minimal discomforts related to the implantation procedure and no need for routine external catheter care. Most importantly, the quality of the patient's life is dramatically improved without external tubes. For these reasons, we believe that vascular access devices should be indicated for patients with malignant tumors as much as placement of these devices is technically feasible.     

In Vitro and In Vivo Characterization of a Silk Fibroin‐Coated Polyester Vascular Prosthesis

Silk fibroin (SF) is well known to be biocompatible, degradable, and nontoxic. In this study, SF was impregnated into a porous polyester graft (InterVascular external velour, InterVascular, Inc., La Ciotat, France), 8 mm in diameter. The SF‐impregnated graft was investigated in vitro and in vivo to evaluate its potential for use as a new vascular graft impervious to blood, while retaining high porosity for tissue ingrowth and biological healing. For in vitro investigation, the water permeability, coating weight, morphology, and mechanical properties of the SF‐impregnated grafts were compared with collagen‐coated grafts (InterGard grafts, InterVascular, Inc.). The water permeability of the controls (1388 ± 30.5 mL/cm²/min at 120 mm Hg) was reduced >99% by SF impregnation, rendering the graft impervious to blood. The coating weight of the collagen was 117 ± 22 mg/g of graft, producing a slightly lower value than the InterGard prosthesis (302 ± 23 mg/g). For the in vivo experiment, six SF‐sealed vascular grafts were implanted in the abdominal aorta of dogs for scheduled periods ranging from 4 h to 6 months. Commercial collagen‐impregnated grafts (InterGard) and untreated external velour grafts (InterVascular) were also implanted for scheduled periods ranging from 1 to 6 months for comparison. Gross observation of the explanted grafts and histological examination of the representative sections were conducted for two types of grafts using a light microscope after hematoxylin–eosin staining. These SF‐impregnated grafts showed less foreign body and inflammation reactions, and the SF layer was almost completely absorbed. The average of the values in each period for the SF grafts was 48% neointima at 1 month, 85% at 3 months, and 97% at 6 months, whereas those of the InterGard prostheses was 34, 46, and 90%, respectively. This study demonstrated that the use of a biodegradable SF as biological sealant can be a feasible approach to prepare impervious textile arterial prostheses. The SF‐impregnated graft showed less thrombogenesis and induced host cell migration along the matrix without foreign body or inflammatory reactions. Moreover, it appears to facilitate the development of endothelial‐like cells.     

Improved retention of endothelial cells seeded on polyurethane small-diameter vascular grafts modified by a recombinant RGD-containing protein

Sponge-type small-diameter vascular grafts were fabricated from a medical-grade polyurethane, Pellethane 2363-80A, by utilization of a salt casting technique. The grafts were compliance matched with a storage modulus of 0.53 +/- 0.08 MPa. The luminal surface of grafts was modified with a thin layer ( approximately 40 micro m) of gelatin crosslinked by epoxide. Then a special Arg-Gly-Asp (RGD)-containing recombinant protein, named CBD-RGD (cellulose binding domain RGD-containing protein), was coated onto the gelatin layer. The platelet adhesion and activation on such a gelatin/CBD-RGD modified surface was significantly reduced. Human umbilical vein endothelial cells were seeded more efficiently onto the modified grafts. There was also a substantial reduction in the subsequent loss of cells from the graft surface following perfusion in vitro. The cell number retained on the modified graft was enhanced by three times after 1 h of perfusion, and by eight times after 3 h of perfusion (retention rate approximately 63%). The retention after 3 h of perfusion could be further increased to nearly 100% if the lined endothelium on gelatin/CBD-RGD modified graft was cultured for another week before perfusion. The modified surface was also shown to help canine external jugular vein endothelial cells to maintain the round cell morphology in vitro.