Endothelial cell attachment and shear response on biomimetic polymer-coated vascular grafts

Endothelial cell (EC) adhesion, shear retention, morphology, and hemostatic gene expression on fibronectin (FN) and RGD fluorosurfactant polymer (FSP)-coated expanded polytetrafluoroethylene grafts were investigated using an in vitro perfusion system. ECs were sodded on both types of grafts and exposed to 8 dyn/cm(2) of shear stress. After 24 h, the EC retention on RGD-FSP-coated grafts was 59 ± 14%, which is statistically higher than the 36 ± 11% retention measured on FN grafts (p < 0.02). Additionally, ECs on RGD-FSP exhibited a more spread morphology and oriented in the direction of shear stress, as demonstrated by actin fiber staining. This spread morphology has been observed earlier in cells that are adapting to shear stress. Real-time PCR for vascular cell adhesion molecule 1, tissue factor, tissue plasminogen activator, and inducible nitric oxide synthase indicated that the RGD-FSP material did not activate the cells and that shear stress appears to induce a more vasoprotective phenotype, as shown by a significant decrease in VCAM-1 expression, compared with sodded grafts. RGD-FSP-coating allows for a cell layer that is more resistant to physiological shear stress, as shown by the increased cell retention over FN. This shear stable EC layer is necessary for in vivo endothelialization of the graft material, which shows promise to increase the patency of synthetic small diameter vascular grafts.     

Role of actin filaments in endothelial cell-cell adhesion and membrane stability under fluid shear stress

Clostridium botulinum C2 toxin (C2 toxin) and purified ADP-ribosylated-alpha-actin (ADP-r-alpha-actin) cause specific actin depolymerisation in living cells. This effect was used to investigate the actin microfilament system with particular emphasis on cell-cell adhesion and plasma membrane integrity in endothelial cells. C2 toxin caused time- and dose-dependent (15-100 ng/ml) changes in endothelial surface morphology (investigated by atomic force microscopy), intercellular gap formation and cell detachment under shear stress. Low concentrations of C2 toxin (1.5 ng/ml), however, did not induce cell detachment but inhibited shear stress-dependent cell alignment. Gap formation as well as cell loss under shear stress was also observed in cells microinjected with purified ADP-r-alpha-actin. Intercellular gap formation was mediated by increased alpha-catenin solubility (40%) due to actin filament depolymerisation. Disintegration of plasma membranes (measured by LDH release) and cell fragmentation during simultaneous exposure to shear stress and C2 toxin were due to a loss of more than 50% of membrane-associated actin. These data show that small disturbances in actin dynamics inhibit shear stress-dependent cell alignment; that depolymerisation of actin filaments increases the solubility of alpha-catenin, thus resulting in cell dissociation and that actin filaments of the membrane cytoskeleton are required to protect the cells from haemodynamic injury such as shear stress. Together, the study shows a heterogeneous regulation of actin filament dynamics at subcellular locations. Junction-associated actin filaments displayed the highest sensitivity whereas stress fibres were far more stable.     

流体切应力梯度对血管内皮细胞排列和形状的影响

目的研究不同梯度切应力作用下,血管内皮细胞(endothelial cells,ECs)排列和形状变化,旨在了解流体切应力梯度对ECs形态的影响,为进一步探讨其功能变化提供实验基础。方法建立可对体外培养ECs施加梯度切应力的流动腔装置,并应用该装置对人脐静脉ECs加载了大小在15dyn/cm2～6.6dyn/cm2(1dyn=10-5N)范围、梯度分别为1.5dyn/cm2和3dyn/cm2的切应力,加载时间均为6h。比较这两种不同切应力梯度对ECs的细胞方向角、细胞宽长比和细胞形态指数的影响。结果在不同切应力梯度作用下,ECs的细胞方向角分布散乱,细胞无排列规律。与3dyn/cm2相比,1.5dyn/cm2切应力梯度下ECs的宽长比和细胞形态指数明显减少,趋向于拉伸状态。结论在不同切应力梯度作用下,ECs均排列紊乱,无规律可循。然而,在相对较小的切应力梯度作用下,细胞容易被拉伸,细胞形状趋向于伸长,而较大切应力梯度作用下,细胞形状则趋向于圆形。     

Gene response in endothelial cells cultured on engineered surfaces is regulated by shear stress

In vitro endothelialization of small-diameter synthetic vascular prostheses confluently lined with cultured autologous endothelial cells (ECs) before implantation has been shown to increase their patency. Many authors have studied the effects of shear stress on EC gene response seeded on various substrates showing different gene expression profiles according to cell type, flow times, or shear type with different molecular biology techniques, but few studies have reported any EC gene response to shear stress when cells are seeded on vascular grafts. The purpose of this in vitro study was to investigate whether ECs were able to transduce shear stress at the level of the nucleus. Human saphenous vein ECs were seeded on glass slides coated with gelatin or fibrin glue or on 6-mm fibrin-glue-coated grafts. Then cells were exposed to 12 dyn/cm(2) for 4 h and ribonucleic acid (RNA) were extracted. The relative messenger RNA (mRNA) expression was studied using real-time quantitative polymerase chain reaction for the following mRNAs: von Willebrand Factor, tissue-plasminogen activator, CD31, vascular endothelial (VE)-cadherin, beta(1) integrin, and vascular endothelial growth factor receptor type 2. From parallel flow chambers, results have shown similar EC gene response on gelatin and fibrin glue under laminar shear stress with downregulation of prothrombotic genes, as well as upregulation of nonthrombotic genes and upregulation of adhesion molecules such as VE-cadherin, but some discrepancies are noted, with a downregulation of CD31 and kinase insert domain receptor (KDR) for the former, without significant variation for the latter. In comparison, results show upregulation of tissue type plasminogen activator gene and downregulation of KDR, VE-cadherin, and beta(1) integrin genes in ECs lining grafts. To conclude, the major finding of our study is to show that human saphenous vein ECs seeded on fibrin glue (in planar flow chambers or in tubular grafts) can be regulated using shear stress via gene expression changes in a nonthrombotic way.     

Fibronectin and F-actin redistribution in cultured endothelial cells exposed to shear stress

Cultured endothelial cells exposed to shear stresses in vitro undergo a reorganization of their F-actin-containing cytoskeletons which culminates in realignment with flow direction. Since a close transmembrane association exists between actin microfilaments and extracellular fibronectin, this study was undertaken to examine whether the actin reorganization induced by shear stress is accompanied by perturbations in the underlying fibronectin matrix. In a closed circulatory loop, bovine endothelial monolayers were exposed to steady, laminar flows corresponding to shear stress levels of 6 and 26 dynes/cm2 for 2, 6, 12, and 24 hours. The co-distribution of fibronectin and F-actin was determined in specimens which were double-labeled with antiserum to fibronectin and rhodamine phalloidin, respectively. Under the influence of shear stress, cells underwent coordinate shape changes resulting in varying degrees of alignment with flow direction. Reorientation at these shear stress levels was dependent on both the time of exposure and the magnitude of shear stress and was accompanied by a reorganization in cellular fibronectin and F-actin. In controls (no flow) correspondence between the two proteins was limited to similarly arranged, radial foci of fibronectin and F-actin filaments at the basal cell surfaces. In flow specimens, coincidence was detected only between occasional fibronectin fibrils and F-actin stress fibers. As a consequence of shear stress, fibronectin became more uniformly distributed beneath monolayers and frequently was organized into bands of densely packed fibrils. Despite this extensive reorganization, rearrangement of fibronectin did not result in the formation of identical, linear structures with F-.     

Endothelialization of PTFE vascular grafts under flow induces significant cell changes

BACKGROUND: To minimize thrombogeneity of small diameter PTFE grafts they are usually coated in vitro with endothelial cells under static culture conditions. The disadvantage of this technique is that a cell layer is formed that fails to withstand shear stress typical in normal blood flow. METHOD: Since the in vivo functional and structural status of endothelial cells correlates with the applied shear stress, we developed a computer-controlled perfusion system to seed and culture cells on PTFE-grafts up to a confluent monolayer under the influence of increasing shear stress. The confluence of endothelial coating was defined by immunohistological staining of cross sections, and by upper light microscopy of flattened graft samples. In addition, the expression of fibronectin as an important adhesion molecule was estimated. RESULTS AND DISCUSSION: The application of pulsatile shear stress (6.6 dyn/cm2, 5 min) to grafts endothelialized under perfusion (n = 7) did not lead to a disruption of the confluent cell layer. In contrast, a 5 min long shear stress of 3 dyn/cm2 was sufficient to wash more than 50% of cells off the PTFE-graft cultured under static conditions (n = 6). The perfusion cultures showed a significantly higher proliferation rate in comparison with static cultures. This effect was reproducibile in both serum-containing and serum-free culture media. The expression of fibronectin by endothelial cells was significantly higher in the perfused graft compared to the static one. These results suggest the practicability of endothelialized PTFE vascular grafts, preconditioned to shear rates similar to the in vivo situation, as an alternative bypass material in cardiac surgery.     

Effect of streptavidin affinity mutants on the integrin-independent adhesion of biotinylated endothelial cells

We have previously shown that the high-affinity streptavidin (SA)-biotin interaction enhanced the initial integrin-mediated adhesion of biotinylated endothelial cells to SA-coated surface by serving as an extrinsic bond to stabilize and enhance the intrinsic fibronectin-integrin binding between the cell and surface. However, the SA-biotin interaction produced considerable detachment by cohesive failure of the membrane. In this study, we examined the hypothesis that reducing the SA-biotin bond affinity could reduce cohesive failure without reducing overall cell detachment. Two mutants of SA, W120F and W120A in which the tryptophan residue at position 120 of the SA molecule was substituted by phenylalanine and alanine, respectively, were characterized and tested in cell adhesion experiments. The binding affinity (K(A)) of SA to adsorbed biotin-labeled bovine serum albumin (b-BSA) ranged from 5.2+/-0.1 x 10(10)M(-1) for wild-type to 3.3+/-0.2 x 10(9)M(-1) for W120F and 4.1+/-1.0 x 10(6)M(-1) for W120A. One hour after cell attachment, the critical shear stress was 26.8+/-2.9 dyn/cm(2) for WT, 26.6+/-3.0 dyn/cm(2) for W120F, and 15.4+/-3.0 dyn/cm(2) for W120A. The focal contact areas of adherent cells were greater for the WT and W120F than the lower affinity mutant, W120A. When shear flow was applied to detach adherent cells, adhesive failure (ligand bond breakage) was favored over cohesive failure (membrane rupture), as the SA binding affinity decreased. Thus, cell adhesion augmented by SA-biotin linkages is dependent on the affinity constants of the SA-biotin bonds, but the reduction in cohesive failure was offset by a reduced strength of adhesion.     

Examination of membrane rupture as a mechanism for mammalian cell detachment from fibronectin-coated biomaterials

Synthetic biomaterials intended for the reconstruction of tissues and organs must be capable of sustaining adhesive contact with adjacent cells and tissues under mechanical and hydrodynamic stresses. To facilitate this adhesion, extracellular matrix proteins or peptide sequences are frequently immobilized to the biomaterial interface. These ligands enhance cell attachment by raising the number of cell receptor/ligand interactions, but consequently they may alter the mechanism of cell detachment. In particular, as the cell membrane is more strongly immobilized to the substratum, the tendency for cell detachment to involve membrane rupture may increase. To test this hypothesis, cells were fluorescent stained with a membrane dye, allowed to attach to fibronectin-coated model substrates for 30 min, and then subjected to a spatially dependent range of shear stress for 5 min (28-220 dyn/cm2) using a radial-flow chamber. Phase-contrast and fluorescent images were analyzed to determine the probability for cell detachment and the area of fluorescent debris left by detaching cells as a function of fibronectin concentration, magnitude of shear stress, and time. It was found at all concentrations of fibronectin that the majority of detaching cells left membrane fragments, the mean size of these fragments was independent of shear stress, and the shape independent of the direction of flow. However, mean fragment area increased with concentration of fibronectin and decreased with duration of shearing flow. We postulate that the area of debris reflects the extent of cell attachment prior to the application of shear and that adhesive complexes can disassemble at the onset of flow.     

Development of novel submicron textured polyether(urethane urea) for decreasing platelet adhesion

Ventricular assist devices have proven to be a useful clinical option for providing circulatory support as a bridge to transplantation and a mode of destination therapy. Thromboembolism is prevented by designing devices that use blood interfaces that either encourage biological material deposition and strong adhesion, or discourage deposition via surface chemistry, surface finish, and fluid flow fields. Minimum continuous or periodic wall shear forces and maximum time at reduced shear are important, and sometimes difficult-to-satisfy, design constraints. We present an approach to reducing platelet adhesion via surface topography, reducing surface area for platelet-material interaction. Large areas of polyether(urethane urea) were textured with two different sizes of ordered pillar arrays via two-stage replication molding without affecting surface chemistry. Pillars had subplatelet dimensions designed to reduce the surface area a platelet may contact. Platelet adhesion was assessed in a physiologically relevant shear stress range from 0-10 dyn/cm2 using a rotating disk and compared to smooth control. Adhesion was highest from 0-5 dyn/cm2. Surface texturing reduced platelet adhesion without increasing platelet activation in bulk suspension. This study demonstrates that material surface texture is an additional variable that may be used to reduce platelet adhesion under low shear stresses potentially reducing thromboembolism.     

Response of mesenchymal stem cells to the biomechanical environment of the endothelium on a flexible tubular silicone substrate

Understanding the response of mesenchymal stem cells (MSCs) to forces in the vasculature is very important in the field of cardiovascular intervention for a number of reasons. These include the development of MSC seeded tissue engineered vascular grafts, targeted or systemic delivery of MSCs in the dynamic environment of the coronary artery and understanding the potential pathological calcifying role of mechanically conditioned multipotent cells already present in the vessel wall. In vivo, cells present in the coronary artery are exposed to the primary biomechanical forces of shear stress, radial stress and hoop stress. To date, many studies have examined the effect of these stresses in isolation, thereby not presenting the complete picture. Therefore, the main aim of this study is to examine the combined role of these stresses on MSC behaviour. To this end, a bioreactor was configured to expose MSCs seeded on flexible silicone substrates to physiological forces - namely, a pulsatile pressure between 40 and 120mmHg (5.33-1.6x10(4)Pa), radial distention of 5% and a shear stress of 10dyn/cm(2) (1Pa) at frequency of 1Hz for up to 24h. Thereafter, the 'pseudovessel' was assessed for changes in morphology, orientation and expression of endothelial and smooth muscle cell (SMC) specific markers. Hematoxylin and eosin (H&E) staining revealed that MSCs exhibit a similar mechanosensitive response to that of endothelial cells (ECs); they reorientate parallel with direction of flow and have adapted their morphology to be similar to that of ECs. However, gene expression results show the cells exhibit greater levels of SMC-associated markers alpha-smooth muscle actin and calponin (p<0.05).     

Endothelial barrier function under laminar fluid shear stress

It has been suggested that increasing levels of shear stress could modify endothelial permeability. This might be critical in venous grafting and in the pathogenesis of certain vascular diseases. We present a novel setup based on impedance spectroscopy that allows online investigation of the transendothelial electrical resistance (TER) under pure laminar shear stress. Shear stress-induced change in TER was associated with changes in cell motility and cell shape as a function of time (morphodynamics) and accompanied by a reorganization of catenins that regulate endothelial adherens junctions. Confluent cultures of porcine pulmonary trunk endothelial cells typically displayed a TER between 6 and 15 ohms cm2 under both resting conditions and low shear stress levels (0.5 dyn/cm2). Raising shear stress to the range of 2 to 50 dyn/cm2 caused a transient 2% to 15% increase in TER within 15 minutes that was accompanied by a reduction in cell motility. Subsequently, TER slowly decreased to a minimum of 20% below the starting value. During this period, acceleration of shape change occurred. In the ensuing period, TER values recovered, reaching control levels within hours and associated with an entire deceleration of shape change. A heterogeneous distribution of alpha-, beta-, and gamma-catenin, main components of the endothelial adherens type junctions, was also observed, indicating a differentiated regulation of shear stress-induced junction rearrangement. Additionally, catenins were partly colocalized with beta-actin at the plasma membrane, indicating migration activity of these subcellular parts. Shear stress, even at peak levels of 50 dyn/cm2, did not cause intercellular gap formation. These data show that endothelial monolayers exposed to increased levels of laminar shear stress respond with a shear stress-dependent regulation of permeability and a reorganization of junction-associated proteins, whereas monolayer integrity remains unaffected.     

Enhanced endothelial cell retention on shear-stressed synthetic vascular grafts precoated with RGD-cross-linked fibrin

Clinical in vitro endothelialization has been shown to increase the patency of synthetic vascular grafts. The shear stress resistance of the cultured autologous endothelium represents a crucial cornerstone of the concept. We investigated whether an enrichment of the precoating matrix with adhesion sites can augment endothelial cell attachment. Adult human saphenous vein endothelial cells (AHSVECs) were seeded confluently ([58 +/- 11] x 10(3) AHSVECs/cm2) onto 10-cm-long ePTFE (expanded polytetrafluorethylene) vascular grafts (n = 24) precoated with commercial clinically approved fibrin gel (Tisseal) containing various concentrations of cross-linked RGD peptide (0.0, 4.0, 8.0, or 16.0 mg of RGD per milliliter of Tisseal fibrinogen component). Endothelialized grafts were postcultivated for 9 days before they were exposed to a pulsatile circulation model mimicking peak physiological shear stress conditions of the femoral artery (12 dyn/cm2; min/max, -60/+28 dyn/cm2). Cell loss after 24 h was quantitatively determined by image analysis of vital stains. Initial 24-h cell loss was 27.2 +/- 1.7% in grafts precoated with the non-RGD-enriched fibrin matrix. In contrast, cell loss was significantly less on fibrin containing 4.0 mg of RGD peptide per milliliter of Tisseal fibrinogen component (13.3 +/- 7.9%; p < 0.05). Cell loss on fibrin containing 8 and 16 mg of RGD per milliliter of Tisseal fibrinogen component was 41.0 +/- 27.4 and 43.0 +/- 23.2% (p > 0.05), respectively. We conclude that low concentrations of RGD peptide cross-linked into commercial fibrin matrices used for clinical in vitro lining of vascular grafts led to significantly increased endothelial cell retention. The failure of higher RGD concentrations to enhance endothelial cell attachment may be explained by competitive binding of endothelial cells to non-cross-linked RGD.     

The use of mild trypsinization conditions in the detachment of endothelial cells to promote subsequent endothelialization on synthetic surfaces

A necessary condition for endothelialization of small diameter grafts is rapid and firm adhesion of endothelial cells upon exposure to flow. To retain integrins on the cell surface, we assessed the effects of trypsin concentration, the duration of trypsin incubation, and trypsin neutralization methods on endothelial cell adhesion. Human umbilical vein endothelial cells which were detached using 0.025% trypsin for 5 min and seeded onto glass pretreated with fibronectin had close to 100% cell retention when shear stresses as high as 200 dyn/cm2 were applied for 2 min. An equivalent level of cell retention was observed on fibronectin coated Teflon-AF for shear stresses up to 60 dyn/cm2 applied for 4h. Using 0.025% trypsin, initial cell spreading and cell surface alpha5beta1 integrins were increased relative to cells treated with 0.5% trypsin. After 1h of attachment, focal adhesions formed when low trypsin concentrations were used but were less evident with high trypsin concentrations. These results showed that low trypsin concentrations produced faster spreading, a higher number of intact integrins, and rapid focal adhesion formation.     

Endothelial cells cultured on engineered vascular grafts are able to transduce shear stress

In vitro endothelialization of small-diameter vascular prostheses confluently lined with cultured autologous endothelial cells (ECs) before clinical implantation improves their patency. Many authors have studied the effects of shear stress on ECs seeded on various substrates showing activation of mitogen-activated protein (MAP) kinases. Very few studies have reported any functional EC response to shear stress when they are seeded on vascular grafts. The purpose of this in vitro study was to investigate whether ECs were able to transduce shear stress. Human saphenous vein ECs were seeded on 6 mm fibrin-glue-coated grafts, then submitted to 15 dyn/cm(2) for 10, 30, and 120 min. Cell lysates were submitted to Western blot analysis to detect phosphorylated ERK 1/2 and p38. ERK 1/2 activation was observed at 10 min (1.6 fold) followed by a lower activation than under static conditions at 30 and 120 min. Shear stress induced a significant increase in p38 phosphorylation (2.5 fold) at 10 and 30 min, decreasing at 120 min. Thus, ECs are able to transduce shear stress in an in vitro model in closed clinical conditions, but the ERK 1/2 and p38 temporal activation profile is different. We provide new insights into the validity of the vascular tissue engineering approach.     

Evaluation of an in vitro endothelialized vascular graft under pulsatile shear stress with a novel radiolabeling procedure

OBJECTIVE: To improve the hemocompatibility of vascular grafts, endothelial cell (EC) seeding of biomaterials prior to implantation is critical. The current in vitro study was designed to investigate such a feasibility on a collagen-coated heparin-bonded graft and to evaluate cell detachment upon pulsatile shear stress. MATERIALS AND METHODS: Endothelial cells (EA-hy-926) were seeded onto grafts. The endothelialization of the grafts was evaluated by the [3H]-thymidine incorporation, scanning electron microscopy (SEM) and histological examinations. After in situ EC radiolabeling with a novel 99mTc technique, the prostheses were exposed to pulsatile shear stress (0.27 N/m2), mimicking the shear rate occurring in a superficial femoral artery, for 3 h in a flow circuit and EC loss quantified by gamma camera detection. RESULTS: Complete EC coverage was achieved after 5 days. Three hours of artificial perfusion resulted in a low EC loss (12.9+/-0.8%, n = 7). SEM shows EC withstanding shear stress in valleys of prosthesis circumvolutions. CONCLUSIONS: These satisfactory results could be explained by the high affinity of EC for heparinized surfaces in addition to cell surface receptors involved in adhesion to collagen.     

层流剪应力对内皮细胞IL-8受体CXCR1表达的影响

为研究内皮细胞在不同时间、不同大小层流剪应力作用下IL-8受体CXCR 1的表达规律,通过体外培养人脐静脉内皮细胞株EA.Hy926细胞,分别施加5.56、10.02、15.27 dyn/cm2三种剪应力,选取剪切1、2、4和8 h等4个时间点进行观测,Western blot检测IL-8受体CXCR 1表达变化。结果表明,在5.56 dyn/cm2剪应力作用下,与静止组相比,CXCR 1表达随作用时间的增加而显著升高(P<0.01),至4h达到最大值,为对照组的2.2倍。在10.02dyn/cm2剪应力作用下,CXCR 1的表达随剪应力作用时间而相对缓慢增加,但仍高于对照组(P<0.05)。在15.27dyn/cm2剪应力作用下,其CXCR 1的表达随时间呈现较显著性降低(P<0.01),当持续作用8 h,其CXCR 1的表达为对照组62.59%。以上结果表明,不同强度、作用时间的层流剪应力参与调节IL-8受体CXCR 1的表达。     

In vivo performance of dual ligand augmented endothelialized expanded polytetrafluoroethylene vascular grafts

In this study, we examined combinations of three approaches to improve the adhesion of endothelial cells (EC) onto expanded polytetrafluoroethylene (ePTFE) vascular grafts placed at the femoral artery of rats: (1) high-affinity receptor-ligand binding of RGD-streptavidin (SA) and biotin to supplement integrin-mediated EC adhesion; (2) cell sodding to pressurize the seeded EC into the interstices of the ePTFE grafts; and (3) longer postseeding attachment time from 1 to 24 h prior to implantation. An in vitro system, which accounts for cell loss due to both graft handling and shear stress, was designed to optimize conditions for in vivo experiments. Results suggest that longer in vitro attachment time enabled the adherent EC to endure mechanical stresses by forming strong adhesions to the underlying extracellular matrix substrates; cell sodding helped to retain the adherent EC by physically docking the cells against the graft interstices; and the SA-biotin interaction enhanced the early attachment of EC but did not lead to better cell retention or reduced surface coverage of blood clot in the current study. Mechanical manipulation of cells during implantation is a limiting factor in maintaining a confluent EC layer on synthetic vascular grafts.     

稳定层流剪应力对内皮细胞骨架调节蛋白VASP表达的影响

为探讨生理强度的稳定层流剪应力对内皮细胞骨架actin相关蛋白VASP特征影响规律，我们采用胰蛋白酶消化法分离人脐静脉内皮细胞(HUVECs)，模拟体内流动环境，建立平行板流动腔模型。利用细胞图像分析系统和ALEXA488—若丹明一次毒蕈环肽双标记法，观察内皮细胞在稳态层流下形态、actin排列变化与VASP分布变化之间的规律。采用Western blot定量动态检测细胞内VASP表达及磷酸化的水平。结果表明，内皮细胞在10dyn／cm^2剪切作用后，随时间细胞逐渐延长，长轴趋于剪应力作用方向排列，细胞与静息态的细胞相比，细胞内骨架沿剪应力方向重组，与此同时VASP表达增强，沿着actin纤维呈点状分布，尤其集中在细胞膜下actin末端区域；Western blot检测显示在剪切后，细胞内VASP出现快速磷酸化，VASP总体表达量增加，2h达高峰后逐渐恢复，8h后再次逐渐升高。以上结果提示血流动力学特性中剪应力引起了细胞胞质内骨架蛋白分子重组，血管内皮细胞形态改变，在此过程中，VASP发挥骨架调节蛋白的作用。     

Biometric surfactant polymers designed for shear-stable endothelialization on biomaterials

We have developed a series of extracellular matrix (ECM)-like biomimetic surfactant polymers to improve endothelial cell adhesion and growth on vascular biomaterials. These polymers provide a single-step procedure for modifying the surface of existing biomaterials and consist of a poly(vinyl amine) (PVAm) backbone with varying ratios of cell-binding peptide (RGD) to carbohydrate (maltose), ranging from 100% RGD:0% maltose to 50% RGD:50% maltose. Three biomimetic surfaces, as well as a fibronectin (FN)-coated glass surface were seeded at confluence with human pulmonary artery endothelial cells (HPAECs) and exposed to shear stresses ranging from 0-40.6 dyn/cm2 for periods of 2 h and 6 h. Surfaces were examined for HPAEC coverage and cytoskeletal arrangement as a function of time and shear stress. In general, after 6 h of shear exposure, EC retention on 100% RGD > FN > 75% RGD > 50% RGD. The 100% RGD surface maintained more than 50% of its initial EC monolayer at low to moderate shear stresses whereas all other surfaces dropped to approximately 40% or less in the same shear stress range. The most stable surface, 100% RGD, showed a significant increase in cytoskeletal organization at all shear stresses greater than 2.5 dyn/cm2. In contrast, there was no real change in cytoskeletal organization on the FN surface, and there was a decrease on the 75% RGD surface over time. These results indicate that increasing surface peptide density can control EC shear stability. Furthermore, improved shear stability increases with increasing peptide density and is related to the EC's ability to reorganize its cytoskeleton.     

流体剪应力对EA.Hy926细胞IL-8受体mRNA表达的影响

为研究EA．Hy926细胞在不同时间、不同大小流体剪应力作用下IL-8受体CXCR1、CXCR2 mRNA的表达规律,通过体外培养人脐静脉内皮细胞株EA．Hy926细胞,分别施加5．56、10．02、15．27 dyn／cm2三种剪应力,选取剪切5 min、10 min、15 min、20 min、25 min和30 min、1、2、4和8 h等10个时间点进行观测,以半定量RT- PCR方法检测IL-8受体mRNA表达变化。结果表明,在5．56 dyn／cm2剪应力作用下,与静止组相比,各时间点CXCR1 mRNA与CXCR2 mRNA表达均显著升高(P<0．05)。在10．02 dyn／cm2剪应力作用下,CXCR1 mRNA表达随时间相对缓慢下降;而CXCR2 mRNA表达在30min出现短暂的升高,然后随着作用时间的延长开始缓慢下降。在15．27 dyn／cm2剪应力作用下,其CXCR1、CXCR2 mRNA的表达随时间呈现较显著性降低(P<0．01),当持续作用4 h以上其CXCR2 mRNA表达极低。以上结果表明,不同强度、作用时间的流体剪应力参与调节IL-8受体表达。