Lymphocyte and granulocyte migration across the endothelial layer of bovine pulmonary artery intimal explants towards lymphocyte conditioned medium

An intravenous infusion of endotoxin into sheep results in accumulation of equal numbers of lymphocytes and granulocytes in the pulmonary microcirculation. The role of the sequestered lymphocytes in acute lung injury is not known. The present study examines whether lymphocyte migration through pulmonary endothelium contributes to endothelial damage and also examines the effect of lymphokines on granulocyte migration. Bovine pulmonary artery intimal explants were mounted in Boyden chambers and conditioned media, prepared from bovine peripheral blood lymphocytes, was used as the chemoattractant. The rate of 51Cr labelled bovine granulocyte lymphocyte migration into intimal explants was determined over a 3 hr incubation period. Permeability changes were assessed by adding trace amounts of 14C-sucrose and 3H-water to the upper well and following their rate of equilibration with the lower well. 6-Keto-PGF1 alpha was measured in the upper well. Lymphocyte conditioned media was found to be chemotactic for both lymphocytes and granulocytes (lymphocyte migration at 60 min: lymphocyte conditioned media = 18.5 +/- 2.3%, mean +/- s.e. RPMI control = 12.5 +/- 1.5; granulocyte migration at 120 min: conditioned media = 36.1 +/- 5.7, RPMI control = 18.2 +/- 3.0). Ultrastructural examination revealed leukocyte migration followed an orderly sequence during which the leukocytes maintained close contact with the adjacent endothelial cells. No structural evidence of endothelial cell damage was seen at any time examined. Granulocyte migration was associated with an increased rate of 14C-sucrose equilibration after 2 hr of incubation (lower well counts/upper well counts at 2 hr, RPMI control = 0.18 +/- 0.02; lymphocyte conditioned medium = 0.30 +/- 0.04) indicating alteration in the endothelial barrier function. Leukocyte migration, particularly lymphocyte migration, was accompanied by a marked increase in prostacyclin accumulation (3 hr: no leukocytes, 188 +/- 17 ng/ml; lymphocytes, 560 +/- 104). These in vitro findings suggest that lymphocytes and lymphokines may be involved in acute lung injury and also that permeability changes associated with granulocyte migration may depend on the chemoattractant.     

Correlation of permeability with the structure of the endothelial layer of pulmonary artery intimal explants

Changes in vascular permeability are associated with structural damage to endothelial cells. These functional and structural changes can be produced experimentally and examined by using intimal explants from bovine pulmonary artery. Correlation of functional with structural changes allows us to dissect the mechanisms responsible for endothelial damage. We have shown that incubation of intimal explants with histamine causes transient formation of interendothelial dilatations and an increased rate of equilibration of tritiated water and [14C]sucrose across the intimal explant. Exposure to endotoxin also causes interendothelial dilatations but the endothelial damage is more severe than that with histamine, and in vivo experiments show a more prolonged increase in pulmonary vascular permeability. Leukocyte migration has also been suggested to result in a decreased barrier function of the endothelial layer. Experiments with the endothelial layer of intimal explants and separated bovine leukocytes suggest that transendothelial migration may depend on the chemotactic stimulus. Neither granulocyte migration toward zymosan-activated plasma nor lymphocyte migration toward lymphocyte-conditioned medium (RPMI in which lymphocytes were incubated with concanavalin A) leads to detectable increases in explant permeability, but granulocyte migration toward lymphocyte-conditioned medium does result in increased equilibration of [14C]sucrose. Finally, a theoretical model has been used to examine the permeability changes seen for the intimal explants exposed to histamine. The model consists of two compartments with radioactive tracers diffusing across a filter of known permeability. Such a model gives good agreement with data obtained in intact sheep, indicating that mathematical models allow quantitative estimates of barrier function in intimal explants that compare favorably with in vivo data.     

Protein kinase C phosphorylates caldesmon77 and vimentin and enhances albumin permeability across cultured bovine pulmonary artery endothelial cell monolayers.

Cytoskeletal protein (CSP) interactions are critical to the contractile response in muscle and non-muscle cells. Current concepts suggest that activation of the contractile apparatus occurs through selective phosphorylation by specific cellular kinase systems. Because the Ca(2+)-phospholipid-dependent protein kinase C (PKC) is involved in the regulation of a number of key endothelial cell responses, the hypothesis that PKC modulates endothelial cell contraction and monolayer permeability was tested. Phorbol myristate acetate (PMA), a direct PKC activator, and alpha-thrombin, a receptor-mediated agonist known to increase endothelial cell permeability, both induced rapid, dose-dependent activation and translocation of PKC in bovine pulmonary artery endothelial cells (BPAEC), as assessed by gamma-[32P]ATP phosphorylation of H1 histone in cellular fractions. This activation was temporally associated with evidence of agonist-mediated endothelial cell contraction as demonstrated by characteristic changes in cellular morphology. Agonist-induced activation of the contractile apparatus was associated with increases in BPAEC monolayer permeability to albumin (approximately 200% increase with 10(-6) MPMA, approximately 400% increase with 10(-8) M alpha-thrombin). To more closely examine the role of PKC in activation of the contractile apparatus, PKC-mediated phosphorylation of two specific CSPs, the actin- and calmodulin-binding protein, caldesmon77, and the intermediate filament protein, vimentin, was assessed. In vitro phosphorylation of both caldesmon and vimentin was demonstrated by addition of exogenous, purified BPAEC PKC to unstimulated BPAEC homogenates, to purified bovine platelet caldesmon77, or to purified smooth muscle caldesmon150. Caldesmon77 and vimentin phosphorylation were observed in intact [32P]-labeled BPAEC monolayers stimulated with either PMA or alpha-thrombin, as detected by immunoprecipitation. In addition, BPAEC pretreatment with the PKC inhibitor, staurosporine, prevented alpha-thrombin- and PMA-induced phosphorylation of both cytoskeletal proteins, attenuated morphologic evidence of contraction, and abolished agonist-induced barrier dysfunction. These results demonstrate that agonist-stimulated PKC activity results in cytoskeletal protein phosphorylation in BPAEC monolayer, an event which occurs in concert with agonist-mediated endothelial cell contraction and resultant barrier dysfunction.     

Culture of bovine pulmonary artery endothelial cells on Gelfoam blocks.

Conventional methods of endothelial cell culture on monolayers and beads require enzymatic digestion, traumatic scraping, or centrifugation to transfer cells to other experimental systems. Gelfoam, a porous gelatin block, not only supports the growth of bovine pulmonary artery endothelial cells but also allows the rapid transfer of cell-laden blocks from one experimental system to another with minimal intervention. This property has been shown to be especially useful for the rapid fixation of endothelial cells for microscopy using standard histologic methods. Histology confirmed that the trabecular nature of the substrate allows endothelial cells to line the interstices of the sponge matrix and grow in a configuration that simulates the appearance of the endothelium in small vessels and capillaries. The inoculation of 1 x 10(5) endothelial cells on 7.5 mg Gelfoam (24 x 8 x 2 mm blocks) was enhanced by fibroblast growth factor and resulted in cell attachment by day 2 with a cell doubling time of 1.7 days. In addition, endothelial cells completely infiltrated 1, 5 and 7.5 mg Gelfoam blocks, as verified by histology. Assays to quantify cell number and protein were easily performed. To facilitate cell counting, the Gelfoam matrix was rapidly removed by the addition of 0.05 mg/ml collagenase, a concentration that interfered minimally with the assay for cellular protein concentration. The data demonstrate that Gelfoam is a suitable support growth matrix for the in vitro culture of bovine pulmonary artery endothelial cells.     

N-乙酰半胱氨酸抗脂多糖诱导的肺动脉损伤的研究

目的 :探讨N 乙酰半胱氨酸 (N acetylcysteine,NAC)减轻脂多糖 (lipopolysaccharide ,LPS)所致的肺损伤及其机制。方法 :应用血管环张力检测技术和扫描电镜方法 ,观察了NAC对LPS引起的肺动脉反应性及肺动脉内皮细胞超微结构变化的影响 ;并测定了肺动脉组织中丙二醛 (malondialhyde ,MDA)、超氧化物歧化酶 (superoxidedismutes ,SOD)及一氧化氮 (nitricoxide,NO)的变化。结果 :LPS(4μg/ml,7h)可降低肺动脉对乙酰胆碱 (ACh)介导的内皮依赖性舒张反应 ,NAC(0 .5mmol/L)可逆转此种反应降低而对正常肺动脉舒缩反应无明显影响 ;NAC可改善LPS引起的肺动脉内皮细胞超微结构损伤并可逆转LPS引起的肺动脉组织中MDA、NO含量增高和SOD活性降低。结论 :NAC可通过抗氧化作用保护肺动脉内皮细胞并增强肺动脉内皮依赖性舒张反应 ,提示此可能是其发挥抗肺动脉压增高从而改善内毒素所致肺损伤的机制之一。     

Trypsin-induced aggregation of bovine pulmonary artery endothelial cells cultured on microcarriers

We studied adherence between 'luminal' surfaces of pulmonary artery endothelial cells by standard aggregometry techniques, widely used for measuring aggregation of platelets and granulocytes. Using suspensions of bovine pulmonary artery endothelial cells cultured on microcarrier beads, in an aggregometer, we found that trypsin caused endothelial aggregation. The aggregation response occurred at trypsin concentrations as low as 0.001%. The degree of trypsin-induced aggregation indicated by the magnitude of the change in light transmission through the endothelial suspensions was related to the trypsin concentration, reaching a maximum level at trypsin concentrations of 0.01%. We conclude that trypsin, even in very low concentrations, causes adherence between 'luminal' surfaces of pulmonary endothelial cells probably because the enzyme destroys cell surface proteins which are necessary to prevent intercellular adherence. The method we describe may be useful for studying cell-cell interactions of endothelium.     

Synchronized oscillations in cytoplasmic free calcium concentration in confluent bradykinin-stimulated bovine pulmonary artery endothelial cell monolayers

Bradykinin-evoked rises in [Ca2+]i were measured in fura-2-loaded bovine pulmonary artery endothelial cell monolayers by dual wavelength excitation fluorimetry. In monolayers seeded thinly and grown to confluence, bradykinin, in the presence of external Ca2+, evoked a rise in [Ca2+]i composed of an initial peak and subsequent oscillating plateau. In the absence of external Ca2+, bradykinin evoked a rise in [Ca2+]i which then returned to the basal value without oscillating. In monolayers seeded near confluent density, the bradykinin-evoked peak in [Ca2+]i was followed by a steady plateau which showed no oscillation. The addition of the phorbol ester, phorbol 12,13-dibutyrate, to a monolayer during bradykinin-evoked oscillations abolished the oscillations and lowered [Ca2+]i partway back toward the basal level. The addition of the protein kinase C inhibitor, H7, did not abolish oscillatory activity, although the frequency of oscillation was reduced. These results indicate that synchronized oscillatory activity can occur in endothelial cell monolayers. It is suggested that these oscillations are dependent on intercellular coupling developed when the cells are grown to confluence and that the mechanism responsible for generating oscillations in [Ca2+]i requires extracellular Ca2+ and involves protein kinase C.     

内源性过氧亚硝基阴离子介导脂多糖导致肺动脉内皮细胞损伤

在培养的牛肺动脉内皮细胞(bovine pulmonary artery endothelial cells,BPAECs)水平上,观察脂多糖(lipopolysaccharide,LPS)对BPAECs诱生过氧亚硝基阴离子(peroxynitrite,ONOO~-)能力及内皮源性ONOO~-在LPS致BPAECs损伤中的作用。结果显示:(1)LPS剂量依赖性地引起BPAECs诱生ONOO~-生成标志物硝基酪氨酸(nitrotyrosine,NT)的荧光强度(即ONOO~-)明显增多,NT阳性细胞数和百分率也明显增多或增高(P<0.05);iNOS选择性抑制剂氨基胍(AG)明显抑制LPS诱生ONOO~-增多(P<0.05),而NT阳性细胞数和百分率分别减少或降低,但无明显差异。(2)在LPS作用下BPAECs培养上清中的MDA含量和LDH活性明显增多和增高,呈现剂量依赖性效应。加AG后MDA含量明显降低(P<0.001),LDH活性呈降低趋势。(3)LPS可诱导BPAECs凋亡明显增多,用EB荧光染色后可见细胞染色质浓集、核变小等凋亡征象。AG可导致LPS引起的BPAECs凋亡明显减少,但仍明显高于溶剂组。LPS可导致BPAECs线粒体呼吸抑制及膜电位下降。上述结果表明,LPS可引起BPAECs生成ONOO~-增多,ONOO~-参与介导LPS所致BPAECs过氧化损伤与细胞凋亡。     

Regulation of Cu,Zn-superoxide dismutase in bovine pulmonary artery endothelial cells.

To evaluate the regulation of endothelial cell Cu,Zn-SOD, we have exposed bovine pulmonary artery endothelial cells in culture to hyperoxia and hypoxia, second messengers or related agonists, hormones, free radical generating systems, endotoxin, and cytokines and have measured Cu,Zn-SOD protein of these cells by an ELISA developed in our laboratory. Control preconfluent and confluent cells in room air contained 196 +/- 18 ng Cu,Zn-SOD/10(6) cells. A23187 (0.33 microM), forskolin (10 microM), isobutylmethylxanthine (0.1 mM), dexamethasone (1 microM), triiodothyronine (1 microM) and retinoic acid (1 microM) failed to alter this level of Cu,Zn-SOD. Exposure to anoxia and hyperoxia both elevated the level approximately 1.5-2.0-fold over 20% oxygen-exposed controls at 48-72 hr. Similarly, exposures to glucose oxidase (0.0075 units/ml), menadione (12.5 microM), xanthine-xanthine oxidase (10 microM, 0.03 units/ml) and H2O2 (0.0005%) increased the level up to two-threefold over controls at 24-48 hr. Lipopolysaccharide, TGF beta 1, TNF alpha, and Il-1 also increased levels of cellular Cu,Zn-SOD, but only in proliferating cells. Il-2, Il-4, interferon-gamma, and GM-CSF had no effect on Cu,Zn-SOD. All treatments that elevated SOD resulted in inhibition of cellular growth, but decreased growth of cells at confluence alone was not associated with increased Cu,Zn-SOD. We propose from these studies that Cu,Zn-SOD of endothelial cells is not under conventional second messenger or hormonal regulation, but that up-regulation of the enzyme is associated with (and perhaps stimulated by) free-radical or oxidant production that also may be influenced by availability of certain cytokines under replicating conditions.     

肿瘤坏死因子致肺微血管内皮细胞单层通透性损伤的实验研究

目的和方法：用针头式滤器检测肿瘤坏死因子（TNF）作用前后及三种药物干预时大鼠肺微血管内皮细胞（RPMVEC）单层通透性的变化,并用免疫组化的方法检测TNF作用前后细胞F－肌动蛋白的改变。结果：TNF作用30min、60min、90min通透系数Kf值较致伤前显著增高;分别加福莫特罗（FOR）、山莨菪碱或霍乱毒素（CTX）干预时Kf值均显著低于TNF组。而TNF作用90min,RPMVEC F-肌动蛋白发生明显解聚：分别加POR、山莨菪硷或CTX干预时F－肌动蛋白无明显变化。结论：TNF诱导RPMVEC单层通透性增高的机制与细胞F－肌动蛋白解聚有关,FOR、山莨菪碱和CTX可能通过抑制F－肌动蛋白解聚而抑制NF诱导的RPMVEC单层通透性增高。     

Endotoxin induction of an inhibitor of plasminogen activator in bovine pulmonary artery endothelial cells

We have examined the effects of bacterial lipopolysaccharide (endotoxin) on the fibrinolytic activity of bovine pulmonary artery endothelial cells. Endotoxin suppressed the net fibrinolytic activity of cell extracts and conditioned media in a dose-dependent manner (threshold dose, 0.1 ng/ml; maximal dose, 10-100 ng/ml). The effects of endotoxin required at least 6 h for expression. Cell extracts and conditioned media contained a 44-kDa urokinase-like plasminogen activator. Media also contained multiple plasminogen activators with molecular masses of 65-75 and 80-100 kDa. Plasminogen activators in extracts and media were unchanged by treatment of cells with endotoxin. Diisopropyl fluorophosphate (DFP) abolished fibrinolytic activity of extracts and conditioned media. DFP-treated samples from endotoxin-treated but not untreated cells inhibited urokinase and tissue plasminogen activator, but not plasmin. Inhibitory activity was lost by incubation at pH 3 or heating to 56 degrees C for 10 min. These treatments did not affect inhibitory activity of fetal bovine serum. Incubation of 125I-urokinase with DFP-treated medium from endotoxin-treated cells produced an inactive complex with an apparent molecular mass of 80-85 kDa. The complex could be detected by chromatography on Sephadex G-100, but not by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These findings suggest that low doses of endotoxin suppress fibrinolytic activity in endothelial cells by stimulating the production or expression of a fast-acting, relatively labile inhibitor of plasminogen activator.     

Induction of cystine transport in bovine pulmonary artery endothelial cells by sodium arsenite.

Sodium arsenite is one of a number of agents reported to induce a 30-34 kDa 'stress' protein in cells. Other agents which induce this stress protein, including diethyl maleate (DEM) and H2O2, have also been reported to be inducers of cystine transport in fibroblasts, macrophages, endothelial cells and other cell types. We have determined that micromolar levels of sodium arsenite increase cystine transport in bovine pulmonary artery endothelial cells (BPAEC), resulting in increases in intracellular glutathione (GSH). The increase in cystine transport appears to be due to stimulation of the synthesis of a protein analogous to the xc- transport system, a sodium-independent system specific for cystine and glutamate. We have determined that this stimulation is maximal between 8-16 h after addition of sodium arsenite and is inhibited by exogenous GSH. Others have reported that synthesis of the 30-34 kDa stress protein is maximal between 2-4 h and returns to baseline by 6-10 h. We conclude that cystine transport may be considered a 'secondary' stress response and is likely to be modulated by sulfhydryl-reactive agents.     

Effects of hypoxia and hyperoxia on proteoglycan production by bovine pulmonary artery endothelial cells

Bovine pulmonary artery endothelial cells in culture were used to assess the influence of oxygen tension on proteoglycan synthesis. Cells exposed to 3% O2 (hypoxia) for 72 h and then labeled with [35S]sulfate for 5 h accumulated significantly less [35S]proteoglycan in medium than cells exposed to 20% O2 (control). This decrease was due primarily to a reduction in heparan sulfate. Cells exposed to 80% O2 (hyperoxia) for 72 h secreted slightly more [35S]proteoglycan into medium than controls. Greater accumulation of chondroitin sulfate was responsible for the increase. The amount of cell-associated proteoglycan did not change significantly in cells cultured in 3% or 80% O2 as compared with control cells cultured in 20% O2. Proteoglycans produced by hypoxia- or hyperoxia-treated cells were found to be similar in size to proteoglycans produced by cells cultured at 20% O2. Glycosaminoglycan sulfation, as measured by ion-exchange chromatography, did not appear to change with varying oxygen tensions. Our results demonstrate that production of proteoglycans secreted by endothelial cells in culture is sensitive to oxygen tension.     

槲皮素对凝血酶所致的内皮细胞损伤的保护作用研究

本实验采用凝血酶损伤内皮细胞模型,观察了槲皮素对内皮细胞结构和功能的影响．结果表明,槲皮素0．5,5和50umol／L明显降低凝血酶刺激的内皮细胞对内皮素（ET）的释放,增加内皮细胞培养液中组织纤溶酶原激活物（t—PA）量,并可改善内皮细胞的超微结构。     

Methods for microcarrier culture of bovine pulmonary artery endothelial cells avoiding the use of enzymes

Enzymes situated along the luminal surface of pulmonary endothelial cells interact with circulating solutes, notably with vasoactive substances, to regulate the hormonal composition of systemic arterial blood. However, it is becoming clear that the range and complexity of reactions occurring at or near the surface of endothelial cells are greater than previously recognized. In addition, evidence indicates that the quality of cell cultures used to define specific endothelial functions must be carefully controlled, together with development of improved understanding of the effects of long-term culture on pulmonary endothelial cells. We have developed new techniques for the culture of pulmonary endothelial cells which avoid exposure to proteolytic enzymes at both the isolation step and during subculture. A combination of mechanical harvest and culture on microcarrier beads has provided a system for the long-term, large-scale culture of pulmonary endothelial cells, features which to a large extent determine the scope of biochemical studies which can be undertaken.     

Central involvement of Rho family GTPases in TNF-alpha-mediated bovine pulmonary endothelial cell apoptosis

In our recent studies, we defined a critical role for increased levels of myosin light chain (MLC) phosphorylation, a regulatory event in the interaction between actin and myosin in TNF-alpha-induced pulmonary endothelial cell actomyosin rearrangement and apoptosis. The Rho GTPase effector, Rho kinase is an important signaling effector governing levels of MLC phosphorylation which contributes to plasma membrane blebbing in several models of apoptosis. In this study, we directly assessed the role of Rho kinase in TNF-alpha-induced endothelial cell microfilament rearrangement and apoptosis. Inhibition of RhoA GTPase activity by the overexpression of dominant negative RhoA attenuates TNF-alpha-triggered stress fiber formation, consistent with Rho activation as a key event in TNF-alpha-induced cytoskeletal rearrangement. Furthermore, pharmacologic inhibition of Rho kinase as well as dominant negative RhoA overexpression dramatically reduced TNF-alpha-induced bovine endothelial apoptosis reflected by nucleosomal fragmentation as well as caspase 7, 3, and 8 activation. These results indicate that Rho kinase-dependent cytoskeletal rearrangement is critical for early apoptotic events, possibly in the assembly of the death-inducing signaling complex leading to initiator and effector caspase activation, and suggest a novel role for Rho GTPases in endothelial cell apoptosis.     

Hypoxia increases the susceptibility of pulmonary artery endothelial cells to hydrogen peroxide injury.

The effect of hypoxia on subsequent susceptibility of porcine pulmonary artery endothelial cells (PAEC) to hydrogen peroxide (H2O2) injury was studied. Preexposure of PAEC to hypoxia for 3 or more h significantly increased susceptibility to subsequent H2O2 challenge. Analysis of the activities of antioxidant enzymes and xanthine oxidase/dehydrogenase suggested that changes in these enzymes in hypoxic PAEC were not responsible for the increased susceptibility. However, hypoxia resulted in significant time-dependent decreases in total glutathione at 12 h or more. The rate of glutathione regeneration in diethylmaleate-treated PAEC and the rate of uptake of cystine and glycine were significantly lower during hypoxia. Hypoxia also caused depletion of ATP and NADPH levels in PAEC, but these did not occur until well after hypoxia-enhanced susceptibility to H2O2 injury was demonstrable. Alterations in glutathione levels and enhanced susceptibility were reversible when hypoxic PAEC were returned to normoxia. These results indicate that hypoxia increased the susceptibility to H2O2 injury by decreasing the ability of PAEC to maintain and regenerate cellular glutathione content in response to H2O2 challenge.     

Dual-phase response of bovine pulmonary artery endothelial cells to agonists which increase free cytoplasmic calcium concentration

The regulation of free cytoplasmic calcium concentration ([Ca2+]i) was studied in bovine pulmonary artery endothelial cells (BPAEC). The cells were seeded on the inner surface of glass cuvettes, grown to confluency and loaded with INDO-1. Using a multiwavelength method for estimation of [Ca2+]i it was shown that in Ca2+ containing medium a rapid rise of [Ca2+]i occurs in response to bradykinin, ATP or thrombin followed by a much slower decrease in free cytoplasmic calcium. Binding of extracellular Ca2+ by EGTA lowered basal [Ca2+]i but had no effect on the rate of agonist-induced [Ca2+]i increase or its absolute amount. In contrast, the kinetics of [Ca2+]i decrease were entirely different. A rapid (less than 0.5 min) decrease in [Ca2+]i to the basal level was observed immediately after the maximum had been achieved. If excess Ca2+ was added to the medium after EGTA, a second [Ca2+]i rise in response to the agonists occurred. The decrease in [Ca2+]i after the second peak was several times slower than the decrease in Ca2+ free medium. It is concluded that Ca2+ entry from the external medium had no effect on the maximal increase in [Ca2+]i but provides a severalfold increase in the duration the endothelial cell responses to the agonists.     

细胞生长因子对冠状动脉内皮细胞增殖与迁移的影响

目的 :观察肝细胞生长因子 (HGF)和血管内皮细胞生长因子 (VEGF)对体外培养牛冠状动脉内皮细胞(BCAEC)增殖、迁移的影响。方法 :分离和培养BCAEC ,设对照组、VEGF组、HGF组。采用四甲基偶氮唑蓝法(MTT)观察细胞增殖 ;倒置显微镜观察培养的血管内皮细胞的迁移。结果 :对照组、VEGF组、HGF组的OD值分别为 0 .2 2± 0 .0 1、0 .40± 0 .1 4、0 .44± 0 .1 5 ;VEGF组、HGF组BCAEC的增殖率分别为 81 .8%± 1 6 .9%、1 0 0 %±2 1 .1 % ;对照组BCAEC迁移不明显 ,而VEGF组和HGF组BCAEC迁移明显。结论 :VEGF、HGF能促进BCAEC增殖、迁移 ,HGF作用强度不亚于VEGF。