A novel, quantitative model for study of endothelial cell migration and sprout formation within three-dimensional collagen matrices

Interactions between migratory endothelial cells (ECs) and surrounding extracellular matrix (ECM) are of central importance to vascular growth. Here, we present a new model of EC migration and morphogenesis within three-dimensional ECM termed "radial invasion of matrix by aggregated cells" (RIMAC). In the RIMAC model, single aggregates of defined numbers of bovine aortic ECs were embedded within small, lenticular gels of type I collagen supported by annuli of nylon mesh. Culture of the gels in nutrient media resulted in quantifiable, reproducible, radial migration of ECs into the collagen. The angiogenic proteins basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) each stimulated migration of ECs in a concentration-dependent manner. In combination, bFGF and VEGF stimulated migration synergistically. In contrast, transforming growth factor-beta1 inhibited migration of ECs. Low concentrations (0.1-0.5 ng/ml) of VEGF induced ECs to form multicellular sprouts, some of which possessed lumen-like spaces. Mitomycin C, an inhibitor of cell proliferation, did not affect the migration of ECs into collagen induced by 0.5 ng/ml VEGF but moderately inhibited migration induced by 5 ng/ml VEGF. Increasing the density (concentration) of the collagen gel inhibited the migration of single ECs and increased the branching and anastomosis of multicellular sprouts. We conclude that the RIMAC model is a highly efficacious assay for the screening of potentially angiogenic and angiostatic compounds and, moreover, is advantageous for mechanistic studies of vascular morphogenesis.     

Stimulatory effects of insulin and insulin-like growth factor I on migration and tube formation by vascular endothelial cells.

The effects of insulin and insulin-like growth factor I (IGF-I) on migration, proliferation and tube-forming activity of endothelial cells were investigated, by using bovine carotid artery endothelial cells. Migration was assayed by a filter membrane technique and tube formation was assayed by a quantitative angiogenesis in vitro model which we have recently developed. In this model, endothelial cells are cultured between two layers of type I collagen gel and become organized into tube-like structures which mimic capillaries in vivo ultrastructurally. Insulin (50-1000 microunits/ml) and IGF-I (10-200 ng/ml) significantly stimulated migration of endothelial cells in a dose-dependent manner with a maximal stimulation of 3.0-fold at 1000 microunits/ml for insulin and 3.8-fold at 200 ng/ml for IGF-I (P less than 0.01). Insulin at concentrations up to 1000 microunits/ml and IGF-I up to 100 ng/ml did not affect proliferation of endothelial cells. When insulin or IGF-I was added in culture medium on collagen gels, tube-forming activity of endothelial cells was markedly stimulated. The specific lengths of tubes significantly increased with the increase in insulin concentration from 25 to 100 microunits/ml (P less than 0.01). At 100 microunits/ml, the stimulation was 1.77-fold (P less than 0.01). IGF-I (1-100 ng/ml) also stimulated the elongation of tubes dose-dependently with a maximal stimulation of 1.96-fold at 100 ng/ml (P less than 0.01). Thus, insulin and IGF-I at pathophysiological concentrations stimulate migration and tube-forming activity of endothelial cells, suggesting that these polypeptides may stimulate repair of endothelial injury in cases such as atherosclerosis and may act as a stimulator of angiogenesis.     

Adipokine resistin promotes in vitro angiogenesis of human endothelial cells

OBJECTIVE: Resistin may be associated with obesity and cardiovascular diseases. However, it is unknown whether resistin directly contributes to angiogenesis. In the present study, we evaluated the effects of resistin on angiogenic potential, including endothelial cell proliferation, migration, and capillary-like tube formation. METHODS: Human coronary artery endothelial cells (HCAECs) were treated with resistin. Cell proliferation was evaluated by [3H]thymidine incorporation and MTS assays. Cell migration was assessed by a modified Boyden chamber assay. Capillary-like tube formation was studied with a Matrigel model. Several gene expression levels were determined by real-time PCR. Activation of mitogen-activated protein kinases (MAPKs) was determined by Bio-Plex luminex analyzer. Basic fibroblast growth factor (bFGF) was used as a control. Human umbilical vein endothelial cells (HUVECs) and human lung microvascular endothelial cells (HMVEC-L) were also included. RESULTS: Resistin induced both endothelial proliferation and migration in a dose- and time-dependent manner with the maximal effect at 40 ng/ml. Both resistin-induced cell proliferation and migration could be effectively blocked by a resistin-neutralization antibody. In addition, resistin promoted capillary-like tube formation of HCAECs on Matrigel. Resistin also significantly upregulated the mRNA expression of vascular endothelial growth factor receptors (VEGFR-1 and VEGFR-2) and matrix metalloproteinases (MMP-1 and MMP-2) at both mRNA and protein levels. Furthermore, transient phosphorylation of ERK1/2 and p38 was observed after the addition of resistin to HCAECs. The resistin-induced cell proliferation and migration were both completely blocked by specific ERK1/2 and p38 inhibitors. CONCLUSIONS: Resistin induces human endothelial cell proliferation and migration, promotes capillary-like tube formation, upregulates the expression of VEGFRs and MMPs, and activates ERK1/2 and p38 pathways. Thus, resistin may play an important role in angiogenesis-associated vascular disorders.     

Angiogenic synergistic effect of basic fibroblast growth factor and vascular endothelial growth factor in an in vitro quantitative microcarrier-based three-dimensional fibrin angiogenesis system

AIM: To develop an in vitro three-dimensional (3-D) angiogenesis system to analyse the capillary sprouts induced in response to the concentration ranges of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) and to quantify their synergistic activity.METHODS: Microcarriers (MCs) coated with human microvascular endothelial cells (HMVECs) were embedded in fibrin gel and cultured in 24-well plates with assay media. The growth factors bFGF, or VEGF, or both were added to the system. The wells (n = 8/group) were digitally photographed and the average length of capillary-like sprouts (ALS) from each microcarrier was quantitated.RESULTS: In aprotinin-stabilized fibrin matrix, human microvascular endothelial cells on the MCs invaded fibrin,forming sprouts and capillary networks with lumina. The angiogenic effects of bFGF or VEGF were dose-clependent in bhe range from 10 to 40 ng/mL. At d 1, 10 ng/mL of bFGF and VEGF induced angiogenesis with an ALS of 32.13±16.6 μm and 43.75±27.92 μm, respectively, which were significantly higher than that of the control (5.88±4.45 μm, P<0.01),and the differences became more significant as the time increased. In addition, the combination of 10 ng/mL of bFGF and VEGF each induced a more significant effect than the summed effects of bFGF (10 ng/mL) alone and VEGF (10 ng/mL) alone when analyzed using SPSS system for general linear model (GLM) (P= 0.011), and bhat also exceeded the effects by 20 ng/mL of either bFGF or VEGF.CONCLUSION: A microcarrier-based in vitro threedimensional angiogenesis model can be developed in fibrin.It offers a unique system for quantitative analysis of angiogenesis. Both bFGF and VEGF exert their angiogenic effects on HMVECs synergistically and in a dose-dependent manner.     

Basic fibroblast growth factor inhibits the anabolic activity of insulin-like growth factor 1 and osteogenic protein 1 in adult human articular chondrocytes

OBJECTIVE: To determine the effects of basic fibroblast growth factor (bFGF) on the chondrocyte anabolic activity promoted by insulin-like growth factor 1 (IGF-1) and osteogenic protein 1 (OP-1). METHODS: Human articular chondrocytes were cultured in alginate beads or as cartilage explants in serum-free medium with or without IGF-1 (100 ng/ml), OP-1 (100 ng/ml), or bFGF (0-100 ng/ml). Cell survival, proliferation, proteoglycan synthesis, and total proteoglycan accumulation were measured after 21 days of culture in alginate beads, and proteoglycan synthesis was measured in explants. RESULTS: Cell survival was not altered by bFGF at any dose, and chondrocyte proliferation was stimulated only at doses above 1 ng/ml. When combined with IGF-1, 1 ng/ml of bFGF stimulated proliferation to 170% of control, but when combined with IGF-1 and OP-1, proliferation increased to 373% of control. Doses of bFGF of 100 ng/ml decreased total proteoglycan levels accumulated per cell by 60% compared with control and also inhibited the ability of IGF-1 or OP-1 to increase proteoglycan production. Likewise, sulfate incorporation in response to IGF-1 and OP-1 alone or together was completely inhibited by 50 ng/ml bFGF in both alginate and explant cultures. CONCLUSION: The anabolic activity of IGF-1 and OP-1, alone and in combination, is significantly inhibited by bFGF. The results suggest that excessive release of bFGF from the cartilage matrix during injury, with loading, or in arthritis could contribute to increased proliferation and reduced anabolic activity in articular cartilage.     

Angiogenesis alteration by defibrotide: implications for its mechanism of action in severe hepatic veno-occlusive disease

Defibrotide (DF) is a mixture of porcine-derived single-stranded phosphodiester oligonucleotides (9-80-mer; average, 50-mer) that has been successfully used to treat severe hepatic veno-occlusive disease (sVOD) with multiorgan failure (MOF) in patients who have received cytotoxic chemotherapy in preparation for bone marrow transplantation. However, its mechanism of action is unknown. Herein, we show that DF and phosphodiester oligonucleotides can bind to heparin-binding proteins (eg, basic fibroblast growth factor [bFGF] but not vascular endothelial growth factor [VEGF] 165) with low nanomolar affinity. This binding occurred in a length- and concentration-dependent manner. DF can mobilize proangiogenic factors such as bFGF from their depot or storage sites on bovine corneal endothelial matrix. However, these molecules do not interfere with high-affinity binding of bFGF to FGFR1 IIIc but can replace heparin as a required cofactor for binding and hence cellular mitogenesis. DF also protects bFGF against digestion by trypsin and chymotrypsin and from air oxidation. In addition, DF binds to collagen I with low nanomolar affinity and can promote human microvascular endothelial cell-1 (HMEC-1) cell mitogenesis and tubular morphogenesis in three-dimensional collagen I gels. Thus, our data suggest that DF may provide a stimulus to the sinusoidal endothelium of a liver that has suffered a severe angiotoxic event, thus helping to ameliorate the clinical sVOD/MOF syndrome.     

Short-term gene expression changes in cartilage explants stimulated with interleukin beta plus glucosamine and chondroitin sulfate

OBJECTIVE: To determine the short-term effects of glucosamine (GLN) and chondroitin sulfate (CS) on expression of genes encoding inflammatory mediators and matrix enzymes in bovine cartilage explants stimulated with interleukin 1 (IL-1). METHODS: Dose-response experiments were conducted for IL-1, GLN, and CS to select concentrations of each optimized for detecting treatment effects on cartilage explants. Based on the dose-response experiments, treatments included fetal bovine serum (FBS) control, 15 ng/ml IL-1, and 15 ng/ml IL-1 with the addition of 10 microg/ml GLN and 20 microg/ml CS. Media were measured for nitric oxide (NO) and prostaglandin E2 (PGE2) while explants were frozen for RNA extraction at 8, 16, and 24 hours. Gene expression relative to FBS control for inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), microsomal PGE synthase-1 (mPGEs1), nuclear factor-kB p65 subunit (NF-kB), matrix metalloproteinase (MMP)-3 and 13, aggrecanase (Agg)-1 and 2, and tissue inhibitor of metalloproteinase-3 (TIMP-3) were assessed by quantitative real-time polymerase chain reaction (RT-PCR). In a separate study using incubation of explants with the same treatments for 48 hours, proteoglycan release was measured with dimethylmethylene blue assay and TIMP-3 protein was evaluated with Western blots. RESULTS: The GLN and CS combination abrogated IL-1-induced gene expression of iNOS, COX-2, mPGEs1, and NF-kB at all timepoints. NO, PGE2, and proteoglycan release were reduced with the combination. The abundance of stimulated MMP-13, Agg-1, and Agg-2 mRNA was repressed, whereas TIMP-3 was upregulated by the combination at all timepoints. The abundance of TIMP-3 protein was increased by the combination relative to IL-1 at 48 hours. CONCLUSION: GLN and CS in combination suppress synthesis and expression of genes encoding inflammatory mediators and proteolytic enzymes while upregulating TIMP-3. This provides a plausible mechanism for the purported mild antiinflammatory and chondroprotective properties of GLN and CS.     

Investigation of chondrocyte hypertrophy and cartilage calcification in a full-depth articular cartilage explants model

Articular cartilage deterioration, which includes cartilage degradation and chondrocyte hypertrophy, is a hallmark of degenerative joint diseases (DJD). Chondrocyte hypertrophy is initiated in the deep layer of the cartilage; thus, a robust explants model for investigation of hypertrophy should include this zone. The aim of this study was to characterize and investigate the hypertrophy-promoting potential of different endogenous factors on an ex vivo articular cartilage model. The full-depth cartilage explants were harvested from bovine femoral condyle and cultured for 13 days in different conditions: 10 ng/ml oncostatin M + 20 ng/ml TNF-α; 100 ng/ml IGF1; 10–100 ng/ml bFGF; 10–100 ng/ml BMP2; 50 μg/ml ascorbic acid in combination with 10 mM β-glycerophosphate; and 20–100 ng/ml triiodothyronine. The cellular activity and morphology, degradation, formation and calcification, and expression level of hypertrophic markers were investigated. The hypertrophic factors tested all induced cellular activity and marked morphological changes starting at day 4, however, not in a synchronized manner. Both cartilage degradation and formation were induced by T3 (P < 0.05). Only T3 had a full hypertrophic gene expression profile (P < 0.05). We developed and characterized a novel model for investigation of chondrocyte hypertrophy. We speculated that this can become an important investigatory tool for investigation of matrix turnover, chondrocyte hypertrophy and cartilage calcification that are associated with DJD pathogenesis.     

Inhibitory effects of docetaxel on expression of VEGF,bFGF and MMPs of LS174T cell

AIM: To study the effects of non-cytotoxic concentrations of docetaxel on some important angiogenic factors of LS174T Cells. METHODS: The non-cytotoxic concentration of docetaxel and the activity of gelatinase were determined with MTT and gelatin zymography respectively, the expression of VEGF(vascular endothelial growth factor), bFGF (basic fibroblast growth factor), MMP (matrix metalloproteinase) 2 and MMP 9 was investigated with RT-PCR and Western blot. RESULTS: The maximum non-cytotoxic concentration of docetaxel on LS174T Cells was 1.0 ng/ml. Compared with the solvent control group, 0.1, 0.5, 1.0 ng/ml of docetaxel could downregulate the expression of VEGF, bFGF, MMP 2 and MMP 9 and suppress the activity of gelatinase. CONCLUSION: Our study suggests that the non-cytotoxic concentrations of docetaxel have strong antiangiogenic activity on LS174T Cells, which suggests docetaxel may be a promising antiangiogenic agent.     

A Novel, Microcarrier-Based in Vitro Assay for Rapid and Reliable Quantification of Three-Dimensional Cell Migration and Angiogenesis

Angiogenesis in situ occurs within the interstitial extracellular matrix. The complexity of currently used three-dimensional in vitro angiogenesis systems makes it difficult to quantify cellular growth and neovessel formation. To overcome this problem we were interested to develop an angiogenesis system which allows rapid and reliable quantification of three-dimensional neovessel formation in vitro. Endothelial cells were seeded on gelatine coated microcarriers (MCs). Cell-coated MCs were suspended in a solution of fibrinogen which was then induced to polymerize by addition of thrombin. By this way, MCs were entrapped in a three-dimensional fibrin matrix. Within a few hours, endothelial cells began to leave their supporting microcarriers and to migrate into the fibrin gel. Without addition of stimulators of angiogenesis, endothelial cells showed incoherent migration into the matrix. In contrast, in response to fibronectin, basic fibroblast growth factor (bFGF), or vascular endothelial growth factor (VEGF), respectively, endothelial cells assembled to form multicellular capillary-like structures occasionally exceeding 1000 μm in length. Each MC gave rise to a limited number of capillaries. A single culture dish contained hundreds of MCs, ensuring that a sufficient number of random samples was present for a reliable statistical evaluation. The angiogenic response could be easily quantified by determination of the average number of capillary-like formations per MC (cap/MC). The capillary count for macrovascular endothelial cells from the bovine pulmonary artery was 0.14 cap/MC when no angiogenic stimulators were contained within the fibrin gel. Addition of 200 μg/ml fibronectin increased capillary formation to 0.63 cap/MC (P < 0.0001) at Day 6. Already after 3 days, addition of bFGF (30 ng/ml) yielded a capillary count of 1.05 and addition of VEGF (100 ng/ml) resulted in 0.91 cap/MC. In contrast, addition of hyaluronic acid stimulated migration of dispersed endothelial cells into the fibrin matrix without leading to significant capillary formation (0.09 cap/MC). Hydrocortisone alone or in combination with heparin led to a significant inhibition of bFGF-stimulated angiogenesis. We thus have developed a convenient angiogenesis in vitro system which allows reliable quantification of capillary formation in a three-dimensional environment. Based on this assay we conclude that apart from proliferation and migration of endothelial cells, angiogenesis additionally requires the assembly of cells to form multicellular capillaries. This process is strongly induced by the extracellular matrix protein fibronectin. Hyaluronic acid, on the other hand, promotes migration but not capillary formation (assembly).     

The interaction of heparin and basic fibroblast growth factor on collagen synthesis in 21-day fetal rat calvariae.

We examined the interactions of the glycosaminoglycan, heparin, and recombinant human basic fibroblast growth factor (bFGF) on collagen synthesis in 21-day fetal rat calvariae. In calvariae treated for 96 h, heparin (25 micrograms/ml) and bFGF (10(-9) M) inhibited collagenase-digestible protein (CDP) labeling by 52 and 60% of control, respectively, and the combination further inhibited CDP labeling. Inhibition of CDP labeling by heparin (25 micrograms/ml) or bFGF (10(-9), 10(-8) M) was similar in the presence or absence of aphidicolin (30 microM) an inhibitor of cell replication. Heparin selectively inhibited CDP labeling in the osteoblast rich central bone but bFGF alone or in combination with heparin inhibited CDP labeling both in the periosteum and central bone. Heparin and bFGF alone decreased steady state levels of alpha 1(I)procollagen messenger RNA (mRNA) at 24 h and the combination further decreased mRNA levels. A high concentration of insulin-like growth factor-1 (IGF-1, 3 x 10(-8) M) reversed the inhibitory effect of heparin on DNA synthesis and CDP labeling. In contrast, IGF-1 could not reverse the inhibitory effects of bFGF on CDP labeling but enhanced the stimulatory effects of bFGF on thymidine incorporation into DNA. We conclude that the inhibitory effects of heparin and bFGF on CDP are independent of effects on cell replication. We further conclude that both heparin and bFGF inhibit collagen synthesis at a pretranslational site since they decreased procollagen mRNA levels in osteoblasts. However, the inhibition of collagen synthesis by heparin and bFGF appears to involve divergent pathways since exogenous IGF-1 could overcome the effect of heparin but not bFGF on collagen synthesis.     

Retinoic acids up-regulate steroidogenic acute regulatory protein gene

Rat osteoprogenitor cells were used to examine the effects of bFGF on DNA synthesis and the expression of osteoblast (OB)-related genes. bFGF, as low as 0.1 ng/ml, stimulated DNA synthesis. bFGF also increased the mRNA level of osteopontin (OP) and decreased that of type I collagen (COL I). When cultures were grown in dexamethasone (DEX) to induce OB lineage commitment, the expression of COL I, alkaline phosphatase (AP) and OP was greatly enhanced. Subsequent incubation with bFGF partially negated the stimulatory effect of DEX on AP and COL I mRNAs. bFGF also inhibited the expression of osteocalcin mRNA in cells grown in 1,25(OH)₂D₃ and DEX. Combined effects of bFGF with IGF-I or PDGF on DNA synthesis and OP expression were examined. bFGF+IGF-I, but not bFGF+PDGF, was more effective than PDGF alone. By comparing cells from adult and old animals, we found that bFGF-induced mitogenic activity was reduced significantly with age. In contrast, the effect of bFGF on the expression of OB genes was not significantly altered by age. These findings suggest that bFGF plays a dual role as a local positive and negative regulator on proliferation and osteogenic lineage expression, respectively, in osteoprogenitor cells, and that the mitogenic activity in response to bFGF was impaired in aging.     

Activation of the mitogen-activated protein kinase cascade is necessary but not sufficient for basic fibroblast growth factor- and epidermal growth factor-stimulated expression of endothelial nitric oxide synthase in ovine fetoplacental artery endothelial cells

Basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and vascular endothelial growth factor (VEGF) may play important roles in the placental vasculature, not only by controlling cell growth and differentiation, but also by mediating production of local vasodilators such as nitric oxide. As the mitogen-activated protein kinase (MAPK) signal cascade has been widely associated with cell growth in response to growth factors, herein we investigate whether bFGF, EGF, and VEGF also stimulate expression of endothelial nitric oxide synthase (eNOS) via activation of the MAPK cascade in ovine fetoplacental artery endothelial cells. The presence of the receptors for all three growth factors was confirmed by both immunocytochemistry and a functional cell proliferation assay. All three growth factors at 10 ng/ml rapidly (<10 min) activated MAPK. This activation was inhibited by PD 98059, a specific MAPK kinase inhibitor. bFGF and EGF, but not VEGF, dose- and time-dependently increased eNOS protein levels. Maximal stimulatory effects of bFGF and EGF on eNOS protein expression were observed at 10 ng/ml for 24 h of treatment and were associated with elevated eNOS messenger RNA. PD 98059 also significantly inhibited bFGF- and EGF-induced increases in eNOS protein expression. Because treatment with all three growth factors resulted in activation of the MAPK cascade, while bFGF and EGF, but not VEGF, increased eNOS expression, we conclude that activation of the MAPK cascade is necessary, but not sufficient, for bFGF- and EGF-induced increases in eNOS protein expression in ovine fetoplacental artery endothelial cells. Thus, additional signaling pathways are implicated in the different controls of eNOS expression and mitogenesis by growth factors.     

Basic fibroblast growth factor inhibits the anabolic activity of insulin‐like growth factor 1 and osteogenic protein 1 in adult human articular chondrocytes

Objective

To determine the effects of basic fibroblast growth factor (bFGF) on the chondrocyte anabolic activity promoted by insulin‐like growth factor 1 (IGF‐1) and osteogenic protein 1 (OP‐1).

Methods

Human articular chondrocytes were cultured in alginate beads or as cartilage explants in serum‐free medium with or without IGF‐1 (100 ng/ml), OP‐1 (100 ng/ml), or bFGF (0–100 ng/ml). Cell survival, proliferation, proteoglycan synthesis, and total proteoglycan accumulation were measured after 21 days of culture in alginate beads, and proteoglycan synthesis was measured in explants.

Results

Cell survival was not altered by bFGF at any dose, and chondrocyte proliferation was stimulated only at doses above 1 ng/ml. When combined with IGF‐1, 1 ng/ml of bFGF stimulated proliferation to 170% of control, but when combined with IGF‐1 and OP‐1, proliferation increased to 373% of control. Doses of bFGF of 100 ng/ml decreased total proteoglycan levels accumulated per cell by 60% compared with control and also inhibited the ability of IGF‐1 or OP‐1 to increase proteoglycan production. Likewise, sulfate incorporation in response to IGF‐1 and OP‐1 alone or together was completely inhibited by 50 ng/ml bFGF in both alginate and explant cultures.

Conclusion

The anabolic activity of IGF‐1 and OP‐1, alone and in combination, is significantly inhibited by bFGF. The results suggest that excessive release of bFGF from the cartilage matrix during injury, with loading, or in arthritis could contribute to increased proliferation and reduced anabolic activity in articular cartilage.

     

碱性成纤维细胞生长因子及相关细胞因子转染对人骨关节炎软骨细胞的作用

目的 探讨腺病毒介导的人碱性成纤维细胞生长因子(bFGF)单独及与白细胞介素-1受体拮抗蛋白(IL-1Ra)和(或)胰岛素样生长因子(IGF)-1共同转染人骨关节炎(OA)软骨细胞后对软骨细胞的影响.方法 采用单独编码人bFGF的重组腺病毒载体或多重组合的重组腺病毒载体转染单层培养的人OA软骨细胞.6 d后分别检测培养上清液中目的 基因表达和糖胺聚糖(GAG)含量.四甲基偶氮唑蓝(MTT)法及流式细胞术分析软骨细胞的增殖及凋亡.甲苯胺蓝染色及Ⅱ型胶原免疫组织化学染色观察软骨细胞基质的合成.免疫印迹法检测Ⅱ型胶原、基质金属蛋白酶(MMP)-3及其抑制剂-1(TIMP-1)的表达.采用单因素方差分析,并进行组间两两比较.结果 各基因转染后,细胞上清液日的基因表达与OA对照组相比明显增高(P<0.05 ). bFGF单独转染可促进软骨细胞增殖,增加Ⅱ型胶原和蛋白多糖的合成(P<0.05).与bFGF单独转染相比,联合IL-1Ra和(或)IGF-1共同转染后,可降低软骨细胞的凋亡率[分别为:(26.1±1.6)%、(19.4±1.0)%、(18.4±1.1)%、(13.9±1.8)%,P<0.05],进一步增加了软骨基质的生物合成(P<0.05).同时,抑制了MMP-3的表达,增加了TIMP-1的表达.结论 腺病毒介导的bFGF转染入OA软骨细胞可促进细胞增殖,增加基质的合成.与IL-1Ra和IGF-1共转染后可发挥协同作用,进一步增加基质合成;同时,抑制了基质的降解.     

沙利度胺对人脐静脉内皮细胞增殖和血管生成的影响

目的 探讨沙利度胺治疗血管发育不良所致消化道出血的机制.方法 体外培养人脐静脉内皮细胞至对数生长期,分为空白对照组、溶剂对照组(二甲基亚砜)和不同浓度(10、20、40、60、80、100μg/ml)沙利度胺组,根据加或不加成纤维细胞生长因子(bFGF,10 ng/ml),共分为16组.刺激72 h后,MTT法检测细胞增殖情况,酶联免疫吸附法和实时定量PCR法测定血管内皮生长因子(VEGF)、肿瘤坏死因子-α(TNF-α)表达.结果 加或不加bFGF刺激,中、高浓度(≥40/μg/ml)沙利度胺均能抑制人脐静脉内皮细胞增殖.未加bFGF刺激时.20μg/ml沙利度胺能明显抑制VEGF表达.加bFGF刺激时,10 μg/ml沙利度胺即能明显抑制VEGF表达.未检出TNF-α表达.结论 体外实验中,沙利度胺能抑制人脐静脉内皮细胞增殖和VEGF表达,从而抑制血管生成,达到治疗血管发育不良所致消化道出血的目的 .     

Substantial inhibition of neo-intimal response to balloon injury in the rat carotid artery using a combination of antibodies to platelet-derived growth factor-BB and basic fibroblast growth factor

Thirty percent of patients undergoing percutaneous transluminal coronary angioplasty develop recurrent disease within a year. This is usually due to the rapid accumulation of intimal smooth muscle cells and extracellular matrix, which causes luminal narrowing, and is probably orchestrated by several mitogenic and chemotactic factors, of which platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) appear to be particularly important. We have investigated the effects of administering a combination of neutralizing antibodies directed against PDGF-BB and bFGF on neo-intima development following balloon catheter injury in the rat carotid artery. Purified sheep anti-PDGF-BB and anti-bFGF immunoglobulins (IgGs) were administered singly and in combination prior to mechanical injury and daily until sacrifice, 8 days later. Plasma titres of exogenous anti-PDGF-BB and anti-bFGF were maintained at levels 10–20-fold higher than those required to neutralise the mitogenic and chemotactic effects of 20 ng/ml of PDGF-BB, or 10 ng/ml bFGF in vitro. Used singly, anti-PDGF IgG treatment was associated with a 47% reduction in intimal thickness and a 59% reduction in intimal:medial area ratio; anti-bFGF IgG administration caused a 53% reduction in intimal thickness, and a 50% reduction in intimal:medial area ratio. Treatment with a combination of these antibodies resulted in a 83.8% reduction in intimal thickness (P<0.05), and a 91% reduction in intimal:medial area ratio (P<0.01). The latter treatment was also associated with a significantly higher intimal cell density (14.2±1.6×10³ nuclei/mm²) compared to animals receiving non-immune IgG (7.8±0.8×10³ nuclei/mm²; P<0.025), although intimal and medial cell proliferation indices were not significantly different between the groups (P>0.05). Our results suggest that in this particular model, PDGF-BB and bFGF are the major factors controlling neointimal hyperplasia, and that these growth factors are operating principally via an effect on smooth muscle cell migration and extracellular matrix protein accumulation.     

Placental growth factor neutralising antibodies give limited anti-angiogenic effects in an in vitro organotypic angiogenesis model

Vascular Endothelial Growth Factor Receptor (VEGFR) mediated signalling drives angiogenesis. This is predominantly attributed to the activity of VEGFR-2 following binding of VEGF-A. Whether other members of the VEGFR and ligand families such as VEGFR-1 and its ligand Placental Growth Factor (PlGF) can also contribute to developmental and pathological angiogenesis is less clear. We explored the function of PlGF in VEGF-A dependent angiogenesis using an in vitro co-culture assay in which endothelial cells are cultured on a fibroblast feeder layer. In the presence of 2% FS MCDB media (containing limited growth factors) in vitro endothelial tube formation is driven by endogenous angiogenic stimuli which are produced by the fibroblast and endothelial cells. Under these conditions independent sequestration of either free VEGF-A or PlGF with polyclonal and monoclonal antibodies inhibited tube formation suggesting that both ligands are required to drive an angiogenic response. Endothelial tube formation could only be driven within this assay by the addition of exogenous VEGF-A, VEGF-E or VEGF-A/PlGF heterodimer, but not by PlGF alone, implying that activation of either VEGFR-2/VEGFR-1 heterodimers or VEGFR-2 homodimers were responsible for eliciting an angiogenic response directly, but not VEGFR-1 homodimers. In contrast to results obtained with an endogenous angiogenic drive, sequestration of PlGF did not affect endothelial tube formation when the assay was driven by 1 ng/ml exogenous VEGF-A. These data suggest that although neutralising PlGF can be shown to reduce endothelial tube formation in vitro, this effect is only observed under restricted culture conditions and is influenced by VEGF-A. Such data questions whether neutralising PlGF would have a therapeutic benefit in vivo in the presence of pathological concentrations of VEGF-A.     

TGF-beta1, -beta2 and -beta3 cooperate to facilitate tubulogenesis in the explanted quail heart

BACKGROUND: Transforming growth factor-beta (TGF-beta) isoforms have been implicated as both pro- and anti-angiogenic modulators. In this study we addressed the roles of TGF-beta isoforms on coronary tubulogenesis. METHODS: Embryonic (E6) quail ventricular specimens were explanted onto collagen gels allowing endothelial cells to migrate and form vascular tubes. Growth factors and/or neutralizing growth factor antibodies were added to the cultures. Endothelial cells were identified using a quail endothelial cell marker, QH1. Image analysis was used to quantify aggregate tube length. RESULTS: Addition of any isoform (TGF-beta(1), TGF-beta(2) or TGF-beta(3)) virtually prevented tubulogenesis (>95% inhibition), while stimulation of tubulogenesis occurred by adding neutralizing antibodies to TGF-beta(3), but not to TGF-beta(1) or -beta(2). When all three isoforms were added, tubulogenesis was enhanced, indicating the key role of TGF-beta(3). Documentation of the inhibitory effect of TGF-beta isoforms on tubulogenesis is further supported by our experiments in which the marked enhancement of tube formation by bFGF and VEGF was negated when exogenous TGF-beta(1), -beta(2), or -beta(3) were added to the cultures. CONCLUSIONS: (1) TGF-beta(1), -beta(2) and -beta(3) each inhibits angiogenesis; (2) cooperation between the three TGF-beta isoforms and other angiogenic factors is essential for the regulation of normal tubulogenesis and (3) the stimulatory effect of VEGF or bFGF on tubulogenesis is negated by exogenous TGB-betas.