Effects of fibrin on the angiogenesis in vitro of bovine endothelial cells in collagen gel

Summary The effect of fibrin on angiogenesis in vitro was investigated using an experimental model of tube formation by bovine capillary endothelial cells (BCEs) in type I collagen gel. One milligram per milliliter of fibrin added into type I collagen gel significantly increased the length of the tubular structures formed by BCEs in the gel by about 180% compared with type I collagen only. The facilitating effect of fibrin on tube formation by BCEs was inhibited by either anti-basic fibroblast growth factor (bFGF) IgG (25 μg/ml) or anti-urokinase type plasminogen activator (uPA) IgG (10 μg/ml) added to the gel and culture medium, but not by anti-tissue type plasminogen activator (10 μg/ml) or non-immune IgG. The Arg-Gly-Asp (RGD) containing peptides (100 μg/ml) added to the culture medium also suppressed tube formation by BCEs in fibrin-containing type I collagen gel, but not in type I collagen gel. These results suggest that the increased release of bFGF and uPA by BCEs therefore plays a role in the angiogenic effect of fibrin in vitro, and the angiogenic effect of fibrin is mediated by the RGD sequence in fibrin, probably via the function of integrin receptor of the BCEs.     

The effect of fibrin on endothelial cell migration in vitro

Endothelial cells are known to migrate and come into contact with fibrin during numerous physiological processes, such as in wound healing and in tumor growth. The present study was initiated to investigate the effect of fibrin on endothelial cell migration in vitro. Endothelial cell migration was assayed by wounding confluent monolayers of bovine aortic endothelial cells with a razor blade and counting the number of cells crossing the wound per unit time. Wound-induced proliferation of endothelial cells was inhibited by mitomycin C-treatment without affecting endothelial cell migration, indicating that in this assay migration could be measured independent of proliferation. Migration of endothelial cells in vitro was inhibited by fibrin in a concentration dependent manner. Endothelial cell migration under fibrin was further reduced by plasminogen depletion of the serum, and fibrin still inhibited the migration of mitomycin C-treated endothelial cells. Kadish et al. (Tissue and Cell, 11, 99, 1979) previously reported that fibrin did not affect EC migration in vitro. The inability to inhibit EC migration with fibrin appears to be due to their assay system which employed agarose, since pre-treating the wounded monolayer with agarose eliminated the inhibition of EC migration by fibrin. The present results indicate that EC migration in vitro can be used as a model system for studying the interaction of fibrin with EC.     

Effect of spatial gradient in fluid shear stress on morphological changes in endothelial cells in response to flow

Arterial bifurcations are common sites for development of cerebral aneurysms. Although this localization of aneurysms suggests that high shear stress (SS) and high spatial SS gradient (SSG) occurring at the bifurcations may be crucial factors for endothelial dysfunction involved in aneurysm formation, the details of the relationship between the hemodynamic environment and endothelial cell (EC) responses remain unclear. In the present study, we sought morphological responses of ECs under high-SS and high-SSG conditions using a T-shaped flow chamber. Confluent ECs were exposed to SS of 2-10 Pa with SSG of up to 34 Pa/mm for 24 and 72 h. ECs exposed to SS without spatial gradient elongated and oriented to the direction of flow at 72 h through different processes depending on the magnitude of SS. In contrast, cells did not exhibit preferred orientation and elongation under the combination of SS and SSG. Unlike cells aligned to the flow by exposure to only SS, development of actin stress fibers was not observed in ECs exposed to SS with SSG. These results indicate that SSG suppresses morphological changes of ECs in response to flow.     

Role of p120-catenin in the morphological changes of endothelial cells exposed to fluid shear stress

p120-Catenin is known to play important roles in cell-cell adhesion stability by binding to cadherin and morphological changes of cells by regulating small RhoGTPase activities. Although the expression and binding states of p120-catenin are thought to dynamically change due to morphological adaptation of endothelial cells (ECs) to fluid shear stress, these dynamics remain to be explored. In the present study, we examined the time course of changes in p120-catenin expression and its binding to vascular endothelial (VE)-cadherin in ECs exposed to shear stress. Human umbilical vein ECs began to change their morphologies at 3-6 h, and became elongated and oriented to the direction of flow at 24 h after exposure to a shear stress of 1.5 Pa. Binding and co-localization of p120-catenin with VE-cadherin at the foci of cell-cell adhesions were retained in ECs during exposure to shear stress, indicating that VE-cadherin was stabilized in the plasma membrane. In contrast, cytoplasmic p120-catenin that was dissociated from VE-cadherin was transiently increased at 3-6 h after the flow onset. These results suggest that the transient increase of cytoplasmic p120-catenin may stimulate RhoGTPase activities and act as a switch for the morphological changes in ECs in response to shear stress.     

Effects of hypoxia on lipolysis in isolated rat myocardial cells

Summary The effect of hypoxia on myocardial lipolysis (glycerol release) was investigated in freshly isolated, calcium-tolerant rat ventricular myocytes. Hypoxia was produced by gassing the incubation medium (Joklik-minimum essential medium, supplemented with 1.2 mM MgSO₄, 1 mM DL-carnitine, 1.5 mM CaCl₂ and 0.6 mM palmitate bound to 0.15 mM fatty acid free bovine serum albumin) with 95% N₂–5% CO,. Control (normoxic) incubations were carried out under air-5% CO₂ atmosphere. Basal glycerol release increased from 46.6 ± 3.0 nmol/106 cells · 30 min in normoxia to 64.5 ± 4.3 nmol/10⁶ cells · 30 min in hypoxia (p < 0.05). Addition of isoprenaline (10 M) resulted in a significant (p < 0.05) stimulation of the glycerol release both in normoxia and in hypoxia, but the enhancement above basal rates was apparently lower in hypoxia (8.7 ± 2.5 nmol/10⁶ cells · 30 min) than in normoxia (12.2 ± 2.7 nmol/106 cells · 30 min). Furthermore, whereas the isoprenaline-induced rise in lipolysis both in normoxia and hypoxia was prevented by inclusion of propranolol (10 M), propranolol did not affect the hypoxia-induced increase in lipolysis. Thus, the above findings suggest that myocardial lipolysis may be stimulated by local non-adrenergic mechanisms during hypoxia.     

Thrombin bound to a fibrin clot confers angiogenic and haemostatic properties on endothelial progenitor cells

Recent data suggest that endothelial progenitor cells (EPCs) are involved in recanalizing venous thrombi. We examined the impact of a fibrin network, and particularly of adsorbed thrombin, on EPCs derived from cord blood CD34(+) cells. Fibrin networks generated in microplates by adding CaCl(2) to platelet-depleted plasma retained adsorbed thrombin at the average concentration of 4.2 nM per well. EPCs expressed high levels of endothelial cell protein C receptor and thrombomodulin, allowing the generation of activated protein C on the fibrin matrix in the presence of exogenous human protein C. The fibrin matrix induced significant EPC proliferation and, when placed in the lower chamber of a Boyden device, strongly enhanced EPC migration. These effects were partly inhibited by hirudin by 41% and 66%, respectively), which suggests that fibrin-adsorbed thrombin interacts with EPCs via the thrombin receptor PAR-1. Finally, spontaneous lysis of the fibrin network, studied by measuring D-dimer release into the supernatant, was inhibited by EPCs but not by control mononuclear cells. Such an effect was associated with a 10-fold increase in plasminogen activator inhibitor-1 (PAI-1) secretion by EPCs cultivated in fibrin matrix. Overall, our data show that EPCs, in addition to their angiogenic potential, have both anticoagulant and antifibrinolytic properties. Thrombin may modulate these properties and contribute to thrombus recanalization by EPCs.     

Autocrine effects of VEGF-D on endothelial cells after transduction with AD-VEGF-D

Endothelial cells in tumor vessels display unusual characteristics in terms of survival and angiogenic properties which result from the increased expression of VEGF-D and its autocrine effect. To evaluate mechanisms by which VEGF-D leads to such abnormal phenotype, we searched for proteins with modified expression in HUVECs enriched in the recombinant mature VEGF-D (VEGFD^ΔNΔC) delivered by adenovirus. Expression of membrane proteins in endothelial cells was characterized by FACS using anti-human IT-Box-135 antibodies. HUVECs transduced with Ad-VEGF-D^ΔNΔC revealed markedly increased expression of proteins involved in adhesion and migration such as (a) integrins (αVβ5, α2β1, α5β1, αMβ2, αLβ2), (b) matrix metalloproteinases (MMP-2, MMP-9, and MMP-14), (c) components of fibrinolytic system (PAI-1, u-PAR), and (d) CD45, CD98, CD147. Interestingly, there also were numerous proteins with significantly reduced expression, particularly among surface exposed membrane proteins. Thus, it can be concluded that to induce proangiogenic phenotype and facilitate migration of HUVECs, VEGF-D^ΔNΔC not only upregulates expression of proteins known to participate in the cell-matrix interactions but also silences some membrane proteins which could interfere with this process.     

Effects of retinol binding protein-4 on vascular endothelial cells

The study was designed to investigate the effect of retinol binding protein (RBP)-4 on the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways, which mediate the effects of insulin in vascular endothelial cells. The effects of RBP4 on nitric oxide (NO) and insulin-stimulated endothelin-1 (ET-1) secretion and on phosphorylation (p) of Akt, endothelial NO synthetase (eNOS), and extracellular signal-regulated kinase (ERK)1/2 were investigated in bovine vascular aortic endothelial cells (BAECs). RBP4 showed an acute vasodilatatory effect on aortic rings of rats within a few minutes. In BAECs, RBP4-treatment for 5 min significantly increased NO production, but inhibited insulin-stimulated ET-1 secretion. RBP4-induced NO production was not inhibited by tetraacetoxymethylester (BAPTA-AM), an intracellular calcium chelator, but was completely abolished by wortmannin, a PI3K inhibitor. RBP4 significantly increased p-Akt and p-eNOS production, and significantly inhibited p-ERK1/2 production. Triciribine, an Akt inhibitor, and wortmannin significantly inhibited RBP4-induced p-Akt and p-eNOS production. Inhibition of Akt1 by small interfering RNA decreased p-eNOS production enhanced by RBP4 in human umbilical vein endothelial cells. In conclusion, RBP4 has a robust acute effect of enhancement of NO production via stimulation of part of the PI3K/Akt/eNOS pathway and inhibition of ERK1/2 phosphorylation and insulin-induced ET-1 secretion, probably in the MAPK pathway, which results in vasodilatation.     

低氧对胚胎干细胞增殖的影响

目的:观察间歇性低氧和持续性低氧对体外培养的胚胎干细胞(ES细胞)增殖的影响.方法:利用细胞记数法和BrdU (5-溴脱氧尿苷)掺入的流式细胞分析检测细胞增殖,并用RT -PCR的方法检测低氧诱导因子(HIF-1a)的表达变化.结果:①将ES细胞分别放在低氧(3%～10% O2)和常氧(20% O2)的环境中培养24 h后,在低氧环境中培养的ES细胞数较常氧组明显减少;②将ES细胞分别给予间歇性低氧刺激(3%～10% O2),每天10 min,连续4 d后,发现3%低氧组较常氧对照组的细胞增殖明显升高.③用RT-PCR方法观察HIF-1a的表达与细胞增殖的关系,发现在常氧环境中培养的ES即有HIF-1a的表达,ES细胞在持续低氧24 h或间歇性低氧(3%～10% O2)刺激4 d后对HIF-1a的表达均无明显影响.结论:间歇性低氧(3% O2)可明显促进体外培养的ES细胞增殖,而持续性低氧抑制ES细胞增殖,间歇性低氧(3% O2)刺激促进ES细胞增殖的机制尚有待于进一步的研究.     

联合应用大鼠血管内皮细胞和胰岛培养对胰岛功能的影响

目的 探讨通过体外共培养胰岛和血管内皮细胞能否改善胰岛的功能.方法 SD 大鼠分离纯化出胰岛细胞,分为两组：A 组胰岛单纯培养组,B 组胰岛和内皮细胞共培养组.从大鼠的胸主动脉分离纯化出血管内皮细胞,胰岛分离纯化后通过AO/PI 染色和胰岛素释放实验来判断两组胰岛的活性.结果 共培养组胰岛在7 d 内维持正常的形态,90﹪的胰岛通过AO/PI 染色显示良好的活性;胰岛素释放实验显示第7 天（2.21 ± 0.21）和第14 天（2.53 ± 0.21）共培养组和单纯培养组（1.94 ± 0.15,1.71 ± 0.19）刺激指数差异有统计学意义（P 〈 0.05）.结论 应用大鼠血管内皮细胞和胰岛共培养能够改善胰岛的存活及分泌功能.     

Oxidized-LDL induce morphological changes and increase stiffness of endothelial cells

There is increasing evidence suggesting that oxidized low-density lipoproteins (ox-LDL) play a critical role in endothelial injury contributing to the age-related physio-pathological process of atherosclerosis. In this study, the effects of native LDL and ox-LDL on the mechanical properties of living human umbilical vein endothelial cells (HUVEC) were investigated by atomic force microscopy (AFM) force measurements. The contribution of filamentous actin (F-actin) and vimentin on cytoskeletal network organization were also examined by fluorescence microscopy. Our results revealed that ox-LDL had an impact on the HUVEC shape by interfering with F-actin and vimentin while native LDL showed no effect. AFM colloidal force measurements on living individual HUVEC were successfully used to measure stiffness of cells exposed to native and ox-LDL. AFM results demonstrated that the cell body became significantly stiffer when cells were exposed for 24 h to ox-LDL while cells exposed for 24 h to native LDL displayed similar rigidity to that of the control cells. Young's moduli of LDL-exposed HUVEC were calculated using two models. This study thus provides quantitative evidence on biomechanical mechanisms related to endothelial cell dysfunction and may give new insight on strategies aiming to protect endothelial function in atherosclerosis.     

高原低氧适应动物新疆灰旱獭右心室重构的组织学改变

目的探讨新疆灰旱獭高原低氧适应性改变致右心室重构组织学改变。方法应用免疫组化技术检测新疆灰旱獭右心室缝隙连接蛋白43(CX43)蛋白表达,同时应用HE染色和Masson染色观察心室肌结构和纤维化程度变化。结果心肌细胞肥大,胶原纤维增多,右心室肥厚指数、体重指数明显增高。CX43蛋白表达减少和(或)分布的改变。结论高原低氧致新疆灰旱獭右心室结构重构,可作为研究高原低氧适应性机制的理想动物模型。     

VEGF189 stimulates endothelial cells proliferation and migration in vitro and up-regulates the expression of Flk-1/KDR mRNA

The vascular endothelial growth factor (VEGF) is a critical factor for development of the vascular system in physiological and pathological angiogenesis. This growth factor exists under at least three isoforms, VEGF120/121, VEGF164/165 and VEGF188/189 which are generated by alternative splicing. VEGF isoforms have different affinities for heparan sulphate as well as for VEGF receptors, and may play distinct roles in vascular development. The role of VEGF189 as an endothelial mitogen, however, remains controversial. VEGF189 is almost entirely bound to the cell surface or extracellular matrix, and is considered active after its cleavage and release from its extracellular binding site. In the present study, we demonstrate that VEGF189 induces endothelial cell proliferation and migration in vitro. The 30-60% increase observed with VEGF189 (10 ng/ml) in HUVEC proliferation was similar to that observed with VEGF165. However, the proliferative effect observed with VEGF189 appeared dependent on the origin of the endothelial cell, since the proliferation was clearly observed with HUVEC but not with BAEC or capillary endothelial cells from dermis (HMEC). The effect of VEGF189 on endothelial cell migration was also analyzed using the wound healing and the Boyden chamber assays. The migration effect was observed with BAEC which do not proliferate with VEGF189, suggesting that different mechanisms are involved in proliferation and migration. In addition, VEGF189 as well as VEGF165 induced a 2-fold increase of Flk-1/KDR expression in HUVEC, the receptor involved in proliferation and migration of endothelial cells. In the Matrigel plug assay in vivo, both VEGF189 and 165 (100 ng/ml) increased the infiltration of endothelial cells. These data suggest that VEGF189 induced endothelial cell migration and proliferation under certain circumstances.     

A fibrin or collagen gel assay for tissue cell chemotaxis: assessment of fibroblast chemotaxis to GRGDSP

Fibroblast chemotaxis is implicated in many physiological processes, including wound healing and morphogenesis. We present a novel assay for chemotaxis of fibroblasts (and other slow-moving tissue cells) in a direct-viewing chamber containing a physiologically relevant three-dimensional fibrin or collagen gel in which long-lasting, spatially continuous gradients have been sustained for at least 24 h, long enough for significant fibroblast migration. This combination of features is not available in any alternative assay of comparable setup simplicity. Using a putative fibroblast chemotactic factor, the fibronectin peptide GRGDSP, we measured human foreskin fibroblast alignment in the direction along the gradient, which followed a biphasic dependence on GRGDSP concentration with an optimal concentration of about 10 nM. Time-lapse video microscopy revealed that cell migration was up the soluble GRGDSP gradient, confirming positive chemotaxis to GRGDSP and rejecting the possibility of dominant haptotaxis down the soluble GRGDSP gradient, that is, up a putative gradient of integrin-mediated adhesion induced by the soluble GRGDSP gradient.     

Tumor skewing of CD34+ cell differentiation from a dendritic cell pathway into endothelial cells

Patients and animals bearing tumors have increased levels of CD34⁺ progenitor cells, which are capable of developing into dendritic cells. However, addition of medium conditioned by murine Lewis lung carcinoma cells increases the cellularity of the CD34⁺ cell cultures and redirects their differentiation into endothelial cells. The resulting cells resemble endothelial cells phenotypically as well as functionally by their capacity to reorganize into cord structures. Mechanisms by which tumors induced the increased cellularity and skewing toward endothelial cells were examined. Tumor-derived VEGF contributed to the increase in cellularity, but not to the redirection of differentiation. Differentiation into endothelial cells was blocked with sTie-2, suggesting tumor-derived angiopoietins in skewing differentiation. These studies show the capacity of tumors to skew progenitor cell development toward endothelial cells and define the mediators that contribute to endothelial cell development.     

In situ positron emission tomography monitoring of endothelial cells embedded in perfused fibrin gels

In tissue engineering, the continuous monitoring of cell and tissue cultures in vitro is crucial to assess their functional status over time. However, these constructs can be large, thick and non-transparent. Medical imaging techniques can allow real-time in situ monitoring of cell and tissue cultures in thick solid scaffolds. Here, human endothelial cells were embedded in fibrin gels that were continuously perfused by a culture medium. Positron emission tomography (PET) imaging was used to assess cell viability non-destructively over periods extending up to a few weeks. PET imaging protocols were adapted and validated to measure culture perfusion and cell metabolism using [¹⁸F]-fluorodeoxyglucose (¹⁸FDG). Cell densities down to 100,000 cells/mL were detectable after 12 h of culture and cell structures were localized within the fibrin gels after 1–2 weeks of culture. PET is a promising tool to investigate a wide range of cellular properties and reveal information on tissue development.     

Effects of fibulin-5 on attachment, adhesion, and proliferation of primary human endothelial cells

BACKGROUND: Fibulin-5 is a novel extracellular protein that is thought to act as a bridging peptide between elastin fibers and cell surface integrins in blood vessel wall. Fibulin-5 binding to endothelial cell (EC) surface integrins may effect cell proliferation and cell attachment to extracellular matrix (ECM) or to artificial surfaces. In this paper, we describe the effects of fibulin-5 on attachment, adhesion, and proliferation of primary human EC. After demonstrating that fibulin-5 over-expression inhibited EC proliferation, we tested the hypothesis that co-expression of fibulin-5 and VEGF165 will lead to unique EC phenotype that will exhibit increased adherence properties and retain its proliferation capacity. METHODS AND RESULTS: Fibulin-5 and VEGF165 gene transfer to primary human saphenous vein endothelial cells was accomplished using retroviral vectors encoding the two genes. Transgene expression was verified using immunohistochemistry, Western blotting, and ELISA. Fibulin 5 over-expression tended to improve immediate EC attachment (30 min after seeding) and improved significantly adhesion (>40%) under shear stress tested 24h after EC seeding. The effects of fibulin-5 and VEGF165 on EC proliferation in the presence or absence of basic FGF were also tested. EC expressing fibulin-5 had reduced proliferation while VEGF165 co-expression ameliorated this effect. CONCLUSION: Fibulin-5 improved EC attachment to artificial surfaces. Dual transfer of fibulin-5 and VEGF165 resulted in EC phenotype with increased adhesion and improved proliferation. This unique EC phenotype can be useful for tissue engineering on endovascular prostheses.     

低氧对CD34~ 造血干/祖细胞增殖分化及其对细胞因子依赖性的影响

采用免疫磁珠法分离脐血CD34 造血干 /祖细胞 ,进行低氧和常氧条件下单个核细胞 (MNC)及CD34 细胞的半固体及液体培养 ,计细胞总数和集落产率 ,并通过流式细胞仪检测细胞表型和细胞周期 ,以探讨造血干/祖细胞在低氧环境下增殖分化性能的改变及其对细胞因子反应性的变化。结果显示 :CD34 细胞在低氧条件下生成的BFU E集落数 ( 32 4 8± 41 4/10 4 细胞 )明显增多 (对照为 191 2± 34 5 /10 4 细胞 ,P <0 0 1) ;在无细胞因子存在的液体培养体系中 ,低氧组的BFU E产率 ( 15 2 4± 2 2 6 /10 4 细胞 )明显高于常氧组 ( 74 2± 9 3/10 4 细胞 ,P <0 0 1) ;低氧培养细胞中CD34 细胞的比例高于对照 2 5± 1 2倍 (P <0 0 5 )。但MNC生成的BFU E在常氧和低氧条件下无显著差异。这些结果表明 :体外低氧环境能显著增加CD34 造血干 /祖细胞形成红系祖细胞的产率 ,且使其对细胞因子的依赖性降低 ,并对早期红系祖细胞的维持有增强作用 ,但对粒系祖细胞的增殖则有抑制作用