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.     

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.     

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.     

Effects of an endothelial cell-conditioned medium on the hematopoietic and endothelial differentiation of embryonic stem cells

We have examined the effect of mouse bone marrow endothelial cell-conditioned medium (mEC-CM) on hematopoietic and endothelial differentiation of mouse embryonic stem cells (mESCs). mEC-CM can efficiently promote the differentiation of mESCs into Flk⁺ cells and hematopoietic colony-forming cells. mEC-CM proved to be as potent as a cytokine cocktail comprised of VEGF, bFGF, IGF and EGF. After inducing mESCs with mEC-CM, cobblestone-like cells were mechanically selected and identified which had the ability to incorporate DiI-Ac-LDL. DiI-Ac-LDL-positive cells were endothelial-like cells due to their expression of CD31 and Flk1, ability to bind to UEA1 and capacity to form capillary-like tube structures on matrigel. In conclusion, mEC-CM can efficiently promote the differentiation of mESCs into endothelial cells and hematopoietic colony-forming cells. The differentiated endothelial-like cells can be isolated by using DiI-Ac-LDL labeling and mechanical selection.     

Effects of shear stress on differentiation of stem cells into endothelial cells

Stem cell transplantation is an appealing potential therapy for vascular diseases and an indispensable key step in vascular tissue engineering. Substantial effort has been made to differentiate stem cells toward vascular cell phenotypes, including endothelial cells (ECs) and smooth muscle cells. The microenvironment of vascular cells not only contains biochemical factors that influence differentiation but also exerts hemodynamic forces, such as shear stress and cyclic strain. More recently, studies have shown that shear stress can influence the differentiation of stem cells toward ECs. A deep understanding of the responses and underlying mechanisms involved in this process is essential for clinical translation. This review highlights current data supporting the role of shear stress in stem cell differentiation into ECs. Potential mechanisms and signaling cascades for transducing shear stress into a biological signal are proposed. Further study of stem cell responses to shear stress will be necessary to apply stem cells for pharmacological applications and cardiovascular implants in the realm of regenerative medicine.     

Using a variant of coverslip hypoxia to visualize tumor cell alterations at increasing distances from an oxygen source

Early stages in tumor development involve growth in confined spaces, where oxygen diffusion is limited and metabolic waste products accumulate. This hostile microenvironment imposes strong selective pressures on tumor cells, leading eventually to the survival and expansion of aggressive subclones that condition further tumor evolution. To model features of this microenvironment in vitro, a diffusional barrier can be introduced in the form of a coverslip placed on top of cells, a method termed coverslip hypoxia. Using a variant of this method, with larger volume between coverslip and cells and with oxygen diffusion occurring only through a small hole in the center of the coverslip, we have visualized alterations in LNCaP tumor cells as a function of their distance to the oxygen source at the center. We observed remarkable morphological changes in LNCaP cells as the distance from the center increases, with cells becoming highly spread, displaying dynamic membrane protrusions and occasionally adopting a migratory phenotype. Concomitantly, cells farther from the center displayed marked increases in the hypoxia marker hypoxyprobe, whereas extracellular pH decreased in the same direction. Cells with altered morphology displayed prominent increases in fibrillar actin, as well as swollen mitochondria with distorted cristae and accumulation of neutral lipid-containing intracellular vesicles. These results show that an in vitro microenvironment that models diffusional barriers encountered by tumors in situ can have profound effects on tumor cells. The coverslip hypoxia variant we describe can be used to characterize in vitro the response of tumor cells to environmental conditions that play crucial roles in early tumor development.     

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。     

肌肽对低氧所致大鼠血管内皮细胞损伤的保护作用

目的：研究肌肽对低氧所致大鼠血管内皮细胞损伤的影响。方法：建立低氧条件下大鼠血管内皮细胞损伤模型,用MTT法观察肌肽对低氧损伤的血管内皮细胞活性的影响,测定细胞培养基中LDH活力,并对细胞骨架进行考马斯亮蓝R-250染色观测其细胞结构。结果：浓度为10mmol/L～20mmol/L肌肽孵育血管内皮细胞6h后,可以抑制缺氧12h和24h引起的血管内皮细胞活性下降,同时减少LDH的释放,保持细胞骨架完整。结论：肌肽对低氧所致的血管内皮细胞损伤具有保护作用。     

Hypoxic preconditioning‐induced autophagy enhances survival of engrafted endothelial progenitor cells in ischaemic limb

Recent clinical studies have suggested that endothelial progenitor cells (EPCs) transplantation provides a modest benefit for treatment of the ischaemic diseases such as limb ischaemia. However, cell‐based therapies have been limited by poor survival of the engrafted cells. This investigation was designed to establish optimal hypoxia preconditioning and evaluate effects of hypoxic preconditioning‐induced autophagy on survival of the engrafted EPCs. Autophagy of CD34⁺VEGFR‐2⁺ EPCs isolated from rat bone marrow increased after treatment with 1% O₂. The number of the apoptotic cells in the hypoxic cells increased significantly after autophagy was inhibited with 3‐methyladenine. According to balance of autophagy and apoptosis, treatment with 1% O₂ for 2 hrs was determined as optimal preconditioning for EPC transplantation. To examine survival of the hypoxic cells, the cells were implanted into the ischaemic pouch of the abdominal wall in rats. The number of the survived cells was greater in the hypoxic group. After the cells loaded with fibrin were transplanted with intramuscular injection, blood perfusion, arteriogenesis and angiogenesis in the ischaemic hindlimb were analysed with laser Doppler‐based perfusion measurement, angiogram and the density of the microvessels in histological sections, respectively. Repair of the ischaemic tissue was improved significantly in the hypoxic preconditioning group. Loading the cells with fibrin has cytoprotective effect on survival of the engrafted cells. These results suggest that activation of autophagy with hypoxic preconditioning is an optimizing strategy for EPC therapy of limb ischaemia.     

CD34+VEGFR‐3+ progenitor cells have a potential to differentiate towards lymphatic endothelial cells

Endothelial progenitor cells (EPCs) play an important role in postnatal neovascularization. However, it is poorly understood whether EPCs contribute to lymphangiogenesis. Here, we assessed differentiation of a novel population of EPCs towards lymphatic endothelial cells and their lymphatic formation. CD34⁺VEGFR‐3⁺ EPCs were isolated from mononuclear cells of human cord blood by fluorescence‐activated cell sorting. These cells expressed CD133 and displayed the phenotype of the endothelial cells. Cell colonies appeared at 7–10 days after incubation. The cells of the colonies grew rapidly and could be repeatedly subcultured. After induction with VEGF‐C for 2 weeks, CD34⁺VEGFR‐3⁺ EPCs could differentiate into lymphatic endothelial cells expressing specific markers 5′‐nucleotidase, LYVE‐1 and Prox‐1. The cells also expressed hyaluronan receptor CD44. The differentiated cells had properties of proliferation, migration and formation of lymphatic capillary‐like structures in three‐dimensional collagen gel and Matrigel. VEGF‐C enhanced VEGFR‐3 mRNA expression. After interfering with VEGFR‐3 siRNA, the effects of VEGF‐C were diminished. These results demonstrate that there is a population of CD34⁺VEGFR‐3⁺ EPCs with lymphatic potential in human cord blood. VEGF‐C/VEGFR‐3 signalling pathway mediates differentiation of CD34⁺VEGFR‐3⁺ EPCs towards lymphatic endothelial cells and lymphangiogenesis. Cord blood‐derived CD34⁺VEGFR‐3⁺ EPCs may be a reliable source in transplantation therapy for lymphatic regenerative diseases.     

The effect of a native collagen gel substratum on the synthesis of collagen by bovine brain capillary endothelial cells

Cultured capillary endothelial cells, derived from bovine brain, and maintained on a plastic substratum synthesized predominantly interstitial collagens of which approximately 75 per cent were secreted into the medium. When grown on a native hydrated collagen type I gel, although no marked alteration in the 'collagen synthetic pattern' was observed, the overall level of collagen synthesis was increased by approximately 100 per cent. More dramatic, however, was the alteration in the distribution of these molecules between medium and cell layer. Interstitial collagens produced by cells grown on collagen gels were almost exclusively associated with the cell layer or collagenous gel. These studies, thus, demonstrate that an extracellular matrix may exert a considerable influence on the cellular synthetic activities and possibly cellular polarity of capillary endothelial cells.     

Injury to primary cultures of rat heart endothelial cells by hypoxia and glucose deprivation

Summary Primary cultures of rat heart endothelial cells were subjected to simulated conditions of ischemia: hypoxia and glucose deprivation for 4 and 24 hr. Cellular injury was evaluated by measuring changes in viability, total protein, cellular morphology, and leakage of cytoplasmic enzymes from the cells into the culture medium. Deprivation of oxygen and glucose for 4 or 24 hr did not lethally injure the cells as noted by no change in cell viability, morphology, and total protein when compared to controls. However, reversible or nonlethal cellular injury was produced as reflected by a significant release of lactate dehydro-genase (LDH) from the cells into the medium after treatment with hypoxia and glucose deprivation for 4 or 24 hr. When the cultures were deprived of glucose, but were oxygenated, cellular injury was not evident after 24 hr. Deprivation of oxygen but not glucose resulted in significant loss of LDH after 4 or 24 hr. When the cultures were allowed to recover after oxygen and glucose deprivation in complete medium containing 1000 mg glucose per l and a normal atmosphere of 20% O₂, they had levels of LDH leakage comparable to those of control cultures. This study was supported by Research Grant HL 18647 from the National Heart, Lung, and Blood Institute and by a National Chicano Council on Higher Education Post-Doctoral Fellowship awarded to D. Acosta from the Ford Foundation. Additional support was provided to D. Acosta by a Faculty Research Assignment Award from the University of Texas Research Institute.