A macrophage factor that stimulates the proliferation of vascular endothelial cells

Sarcoid macrophage-epithelioid cells have been shown to release a growth factor that stimulates the proliferation of vascular endothelial cells in vitro. In the presence of this factor, cultured endothelial cells can proliferate in a serum-free medium. Gel-chromatography on Sephadex G-75 revealed a single peak of activity on endothelial cells. The molecular weight was estimated at 7,000-10,000. The activity was heat-labile and trypsin-sensitive, and did not adhere to heparin-Sepharose.     

Hydrogen peroxide stimulates exocytosis of von Willebrand factor in human umbilical vein endothelial cells

The aim of our research was to study the influence of hydrogen peroxide on the exocytosis of von Willebrand factor (vWF) in human umbilical vein endothelial cells (HUVEC). We have found that H₂O₂ at a non-toxic concentration (100 μM) increases the amount of vWF secreted by HUVEC by 43 ± 14% over control (p < 0.03) and elevates total exposition of vWF on cell surface by 89 ± 5% (p < 0.01). Analysis of immunofluorescent images of HUVEC with CellProfiler program revealed that the average number of antigen positive structures on the single cell surface increases from 11.4 ± 0.16 in control up to 17.5 ± 0.21 after incubation with H₂O₂ (p < 0.01). vWF is exposed on the cell surface as dots with the average sizes around 1–3 μm. H₂O₂ causes an increase in the number of these dots and the appearence of the strings of vWF which are absent in control HUVEC. It is suggested that H₂O₂ may serve as a messenger which stimulates vWF exocytosis.     

Vascular endothelial growth factor stimulates osteoblastic differentiation of cultured human periosteal-derived cells expressing vascular endothelial growth factor receptors

Angiogenesis plays an important role in bone development and postnatal bone fracture repair. Vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptors (VEGFRs) are primarily involved in angiogenesis. This study investigated the expression of VEGF isoforms, VEGFR-1, and VEGFR-2 during the osteoblastic differentiation of cultured human periosteal-derived cells. In addition, the effect of exogenous VEGF on the osteoblastic differentiation of cultured human periosteal-derived cells was also examined. The expression of the VEGF isoforms (VEGF₁₂₁, VEGF₁₆₅, VEGF₁₈₉, and VEGF₂₀₆), VEGFR-1, and VEGFR-2 was observed in the periosteal-derived cells. Administration of KRN633, a VEGFR-1 and VEGFR-2 inhibitor, decreased the alkaline phosphatase (ALP) activity during the osteoblastic differentiation of cultured human periosteal-derived cells. However, the administration of VEGFR2 Kinase Inhibitor IV, a VEGFR-2 inhibitor, did not affect the ALP activity. The addition of recombinant human VEGF₁₆₅ elevated the ALP activity and increased the calcium content in the periosteal-derived cells. Treating the periosteal-derived cells with recombinant human VEGF₁₆₅ resulted in an increase in Runx2 transactivation in the periosteal-derived cells. These results suggest that exogenous VEGF stimulates the osteoblastic differentiation of cultured human periosteal-derived cells and VEGF might act as an autocrine growth factor for the osteoblastic differentiation of cultured human periosteal-derived cells.     

Polyelectrolyte complexes stabilize and controllably release vascular endothelial growth factor

Angiogenesis has long been a desired therapeutic approach to improve clinical outcomes of conditions typified by ischemia. Vascular endothelial growth factor (VEGF) has demonstrated the ability to generate new blood vessels in vivo, but trials using intravenous delivery have not yet produced clinical success. Localized, sustained delivery of VEGF has been proven necessary to generate blood vessels as demonstrated by implantable, controlled release devices. Ultimately, nanoparticles delivered by intravenous injection may be designed to accumulate in target tissues and sustain the local VEGF concentration; however, injectable nanosuspensions that control the release of stabilized VEGF must first be developed. In this study, we utilize the heparin binding domain of VEGF to bind the polyanion dextran sulfate, resulting in an enhanced thermal stability of VEGF. Coacervation of the VEGF-bound dextran sulfate with selected polycations (chitosan, polyethylenimine, or poly-L-lysine) produced nanoparticles approximately 250 nm in diameter with high VEGF encapsulation efficiency (50-85%). Release of VEGF from these formulations persisted for >10 days and maintained high VEGF activity as determined by ELISA and a mitogenic bioassay. Chitosan-dextran sulfate complexes were preferred because of their biodegradability, desirable particle size ( approximately 250 nm), entrapment efficiency ( approximately 85%), controlled release (near linear for 10 days), and mitogenic activity.     

Hydrogen peroxide signaling mediates DHEA-induced vascular endothelial cell proliferation

Dehydroepiandrosterone (DHEA) activates a putative plasma membrane G_i-protein coupled receptor to induce vascular endothelial proliferation. We now test the hypothesis that hydrogen peroxide (H₂O₂) signaling mediates this effect. Incubation of EA.hy926 cells, a human vascular endothelial cell line, with DHEA for 5 min produced a significant increase in H₂O₂ production, measured by oxidation of either p-hydroxyphenylacetate or dichlorodihydrofluorescein. The DHEA effect on H₂O₂ production was maximal at 1 nM DHEA, was evident within the first minute of incubation, and remained for 10 min. Similar results were present in primary bovine aortic endothelial cells. The induction of H₂O₂ in EA.hy926 cells was mimicked by a membrane-impermeable albumin-conjugated DHEA and was inhibited by either catalase or pertussis toxin. Incubation of endothelial cells with DHEA for 5 min resulted in a 2-fold increase of cyclin D1 mRNA and protein expression at 4 h. These effects were abolished by co-incubation with catalase. DHEA induced a 50 ± 7% increase in cell proliferation over 24 h, measured as cellular Ki-67 immunoreactivity. This proliferative effect was abolished by either catalase or pertussis toxin co-incubation, indicating an H₂O₂ and G_i-protein-dependent effect. We conclude that H₂O₂ is a key signaling molecule mediating the proliferative effects of DHEA in vascular endothelial cells, possibly by up-regulating cell-cycle associated genes, such as cyclin D1.     

Cyclooxygenase-2-regulated vascular endothelial growth factor release in gastric fibroblasts

VEGF is a highly specific stimulator of endothelial cells and may play an important role in angiogenesis in the process of tissue regeneration. We previously showed that cyclooxygenase-2 (COX-2) expressed in mesenchymal cells of the ulcer bed is involved in the ulcer repair process. To clarify the role of COX-2 in angiogenesis during gastric ulcer healing, we investigated the relation between COX-2 expression and VEGF production in human gastric fibroblasts in vivo and in vitro. Gastric fibroblasts were cultured in RPMI 1640 with and without IL-1alpha or IL-1beta in the presence or absence of NS-398, a selective COX-2 inhibitor. Supernatant VEGF and PGE(2) concentrations were measured by enzyme-linked immunosorbent assay. COX-2 expression in fibroblasts was determined by Western blot analysis. VEGF and COX-2 expression in surgical resections of human gastric ulcer tissue was examined immunohistochemically. IL-1 dose dependently enhanced VEGF release in cultured gastric fibroblasts after a 24-h stimulation. IL-1 also stimulated PGE(2) production in gastric fibroblasts via COX-2 induction. NS-398 significantly suppressed VEGF and PGE(2) release from IL-1-stimulated gastric fibroblasts; concurrent addition of PGE(2) restored NS-398-inhibited VEGF release. COX-2 and VEGF immunoreactivity were colocalized in fibroblast-like cells in the ulcer bed of gastric tissues. These results suggest that COX-2 plays a key role in VEGF production in gastric fibroblasts stimulated by IL-1 in vitro and that angiogenesis induced by the COX-2-VEGF pathway might be involved in gastric ulcer healing.     

Leptin-induced transphosphorylation of vascular endothelial growth factor receptor increases Notch and stimulates endothelial cell angiogenic transformation

Leptin increases vascular endothelial growth factor (VEGF), VEGF receptor-2 (VEGFR-2), and Notch expression in cancer cells, and transphosphorylates VEGFR-2 in endothelial cells. However, the mechanisms involved in leptin’s actions in endothelial cells are not completely known. Here we investigated whether a leptin-VEGFR-Notch axis is involved in these leptin’s actions. To this end, human umbilical vein and porcine aortic endothelial cells (wild type and genetically modified to overexpress VEGFR-1 or -2) were cultured in the absence of VEGF and treated with leptin and inhibitors of Notch (gamma-secretase inhibitors: DAPT and S2188, and silencing RNA), VEGFR (kinase inhibitor: SU5416, and silencing RNA) and leptin receptor, OB-R (pegylated leptin peptide receptor antagonist 2: PEG-LPrA2). Interestingly, in the absence of VEGF, leptin induced the expression of several components of Notch signaling pathway in endothelial cells. Inhibition of VEGFR and Notch signaling significantly decreased leptin-induced S-phase progression, proliferation, and tube formation in endothelial cells. Moreover, leptin/OB-R induced transphosphorylation of VEGFR-1 and VEGFR-2 was essential for leptin’s effects. These results unveil for the first time a novel mechanism by which leptin could induce angiogenic features via upregulation/trans-activation of VEGFR and downstream expression/activation of Notch in endothelial cells. Thus, high levels of leptin found in overweight and obese patients might lead to increased angiogenesis by activating VEGFR-Notch signaling crosstalk in endothelial cells. These observations might be highly relevant for obese patients with cancer, where leptin/VEGFR/Notch crosstalk could play an important role in cancer growth, and could be a new target for the control of tumor angiogenesis.     

Adrenomedullin stimulates angiogenic response in cultured human vascular endothelial cells: involvement of the vascular endothelial growth factor receptor 2

In recent years, evidence has accumulated that many endogenous peptides play an important regulatory role in angiogenesis by modulating endothelial cell behavior. Adrenomedullin (AM), one such factor, was previously shown to exert a clearcut proangiogenic effect in vitro when tested on specialized human endothelial cells, such as HUVECs and immortalized endothelial cell lines. In the present study we used normal adult vascular endothelial cells isolated from human saphenous vein to analyze in vitro the role of AM, related to both early (increased cell proliferation) and late (differentiation and self-organization into capillary-like structures) angiogenic events and their relationship with the vascular endothelial growth factor (VEGF) signaling cascade. The results indicated that also in this endothelial cell phenotype AM promoted cell proliferation and differentiation into cord-like structures. These actions resulted specific and were mediated by the binding of AM to its AM1 (CRLR/RAMP2) receptor. Neither the administration of a VEGF receptor 2 (VEGFR-2) antagonist nor the downregulation of VEGF production by gene silencing were able to suppress the proangiogenic effect of AM. However, when the experiments were performed in the presence of SU5416 (a selective inhibitor of the VEGFR-2 receptor at the level of the intra-cellular tyrosine kinase domain) the proangiogenic effect of AM was abolished. This result suggests that in vascular endothelial cells the binding of AM to its AM1 receptor could trigger a transactivation of the VEGFR-2 receptor, leading to a signaling cascade inducing proangiogenic events in the cells.     

Transforming growth factor alpha stimulates prostacyclin production by cultured human vascular endothelial cells more potently than epidermal growth factor

Transforming growth factor alpha (TGF alpha) induces dose- and time-dependent stimulation of prostacyclin (PGI2) production by cultured human umbilical vein endothelial cells. The lowest stimulatory concentration of TGF alpha was 0.1 ng/ml and the maximal response, a 2.7-fold rise, was obtained with 10 ng/ml. The stimulation, which lasted at least 24 h, was blocked by cycloheximide and by indomethacin. TGF alpha induced PGI2 production at 10-100 times lower concentrations than did epidermal growth factor (EGF), although in stimulating endothelial cell growth the two factors were equipotent. This is the first demonstration that TGF alpha enhances PGI2 production by human cells. Moreover, this is the first evidence that it acts as both an agonist (growth) and a superagonist (PGI2 production) of EGF in the same cell type. I suggest that this phenomenon may be involved with the angiogenic activity of TGF alpha.     

Hepatitis C virus stabilizes hypoxia-inducible factor 1alpha and stimulates the synthesis of vascular endothelial growth factor

Hepatitis C virus (HCV) infection is one of the major causes of chronic hepatitis, liver cirrhosis, which subsequently leads to hepatocellular carcinoma (HCC). The overexpression of the angiogenic factors has been demonstrated in HCC. In this study, we investigated the potential of HCV gene expression in inducing angiogenesis. Our results show that HCV infection leads to the stabilization of hypoxia-inducible factor 1alpha (HIF-1alpha). We further show that this stabilization was mediated via oxidative stress induced by HCV gene expression. The activation of NF-kappaB, STAT-3, PI3-K/AkT, and p42/44 mitogen-activated protein kinase was necessary for HIF-1alpha stabilization. HIF-1alpha induction in turn led to the stimulation of vascular endothelial growth factor. By using the chick chorioallantoic membrane assay, we show that HCV-infected cells released angiogenic cytokines, leading to neovascularization in vivo. These results indicate the potential of HCV gene expression in angiogenesis.     

p38丝裂原素激活的蛋白激酶在调节低氧诱导人内皮细胞分泌血管内皮生长因子过程中的作用

血管内皮细胞中血管内皮生长因子(vascular endothelial growthfactor,VEGF)的合成增加在促进血管新生的过程中起着非常重要的作用.然而低氧诱导VEGF分泌的细胞内信号转导机制还不是很清楚.人脐静脉内皮细胞系(ECV304)在低氧或常氧的状态下培养12～24 h后分别用实时定量PCR和Western blot的方法来检测VEGF mRNA的表达及ERK1/2和p38激酶的磷酸化水平.分泌到培养液中的VEGF蛋白用酶联免疫吸附(ELISA)的方法来检测.业已报道,ERK的抑制剂PD98059能够抑制低氧诱导的VEGF基因的表达,根据这个报道,我们发现在低氧情况下,ECV304细胞的ERK1/2磷酸化水平增高以及VEGF的合成增加等这些变化也能被PD98059所抑制.本次实验的新发现是p38激酶的激活在低氧诱导VEGF合成增加中的作用.p38激酶的抑制剂SB202190能抑制低氧诱导的VEGF合成增加.这些数据首次直接证实了p38激酶在低氧诱导人内皮细胞分泌VEGF增加过程中的作用.     

Purified scatter factor stimulates epithelial and vascular endothelial cell migration

Fibroblasts and smooth muscle cells release a protein activity which causes epithelial sheets to "scatter" into isolated cells. Purification of scatter factor (SF) activity from ras-transformed 3T3 cells was reported recently. We purified ras-3T3 SF by a slightly different method with essentially similar findings. Purified factor showed a single band at 77 +/- 3 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. Scatter activity was eluted from gel slices at this molecular size. Reduction with mercaptoethanol caused the loss of activity and the appearance of two bands (58 and 31 kDa). We report the amino acid composition of ras-3T3 SF and sequences of several tryptic peptides. These sequences were not similar to the known proteins in the Protein Database. We have shown previously that partially purified ras-3T3 scatter activity stimulates migration of epithelial and vascular endothelial cells in a new migration assay utilizing microcarrier beads. We now demonstrate that the same purified ras-3T3 protein scatters epithelial cells and stimulates epithelial and endothelial migration in microcarrier bead and Boyden chamber assays. Partially purified human smooth muscle scatter activity shares these activities, but the protein(s) responsible has not been isolated. Migration-stimulating activity was maximal at ras-3T3 protein concentrations less than 10 ng/ml (0.13 nM). ras-3T3 SF had no collagenolytic activity and did not stimulate DNA synthesis in fibroblast growth factor-responsive human melanocytes. ras-3T3 SF appears to be a new protein which regulates endothelial and epithelial mobility; and, therefore, it may be involved in vascular repair and wound healing.     

Nicotine-induced vascular endothelial growth factor release via the EGFR-ERK pathway in rat vascular smooth muscle cells

Cigarette smoke has been firmly established as an independent risk factor for atherosclerosis and other vascular diseases. The proliferation and migration of vascular smooth muscle cells (VSMC) induced by growth factors have been proposed to play an important role in the progression of atherosclerosis. In the present study, we investigated the effects of nicotine, which is one of the important constituents of cigarette smoke, on vascular endothelial growth factor (VEGF) release, in rat VSMC. The stimulation of cells with nicotine resulted in a time- and concentration-dependent release of VEGF. Hexamethonium, an antagonist of nicotinic acetylcholine receptor (nAChR), inhibited nicotine-induced VEGF release. We next investigated the mechanisms by which nicotine induces VEGF release in the cells. The nicotine-induced VEGF release was inhibited by treatment with U0126, a selective inhibitor of MEK, which attenuated the nicotine-induced ERK phosphorylation. Nicotine induced a transient phosphorylation of ERK. Furthermore, AG1478, a selective inhibitor of epidermal growth factor receptor (EGFR) kinase, inhibited nicotine-induced ERK phosphorylation and VEGF release. These data suggest that nicotine releases VEGF through nAChR in VSMC. Moreover, VEGF release induced by nicotine is mediated by an EGFR-ERK pathway in VSMC. VEGF may contribute to the risk of cardiovascular diseases in cigarette smokers.     

Hydrogen peroxide stimulates tetrahydrobiopterin synthesis through the induction of GTP-cyclohydrolase I and increases nitric oxide synthase activity in vascular endothelial cells

Tetrahydrobiopterin (BH4), which is an essential cofactor for nitric oxide synthase (NOS), is generally accepted as an important molecular target for oxidative stress. This study examined whether hydrogen peroxide (H(2)O(2)), one of the reactive oxygen species (ROS), affects the BH4 level in vascular endothelial cells (ECs). Interestingly, the addition of H(2)O(2) to ECs markedly increased the BH4 level, but not its oxidized forms. The H(2)O(2)-induced increase in the BH4 level was blocked by the inhibitor of GTP-cyclohydrolase I (GTPCH), which is the rate-limiting enzyme of BH4 synthesis. Moreover, H(2)O(2) induced the expression of GTPCH mRNA, and the inhibitors of protein synthesis blocked the H(2)O(2)-induced increase in the BH4 level. The expression of the inducible isoform of NOS (iNOS) was slightly induced by the treatment with H(2)O(2). Additionally, the L-citrulline formation from L-arginine, which is the marker for NO synthesis, was stimulated by the treatment with H(2)O(2), and the H(2)O(2)-induced L-citrulline formation was strongly attenuated by NOS or GTPCH inhibitor. These results suggest that H(2)O(2) induces BH4 synthesis via the induction of GTPCH, and the increased BH4 is coupled with NO production by coinduced iNOS. H(2)O(2) appears to be one of the important signaling molecules to regulate the BH4-NOS system.     

血管内皮细胞生长因子在非血管新生方面的重要功能

血管内皮细胞生长因子(vascular endothelial growth factor,VEGF或VEGF-A),又称为血管通透因子(vascular permeable factor,VPF)是一种具有多种功能的生物大分子,它是分泌性糖蛋白生长因子超家族中的一员.VEGF主要通过两个高亲和力的酪氨酸激酶受体来传递各种信号:VEGF受体1和2(VEGFR1,VEGFR2),从而引起细胞的多种生理反应.在胚胎时期,VEGF可以促进血管内皮细胞的增殖、迁移、管状形成和提高内皮细胞的存活率,对于血管新生和发育十分关键;而在成体时期,VEGF则主要参与正常血管结构的维持,并调节生理和病理性血管新生.近几年来的临床试验表明,使用多种阻断VEGF作用的抑制剂能有效促进肿瘤血管的退化和减小肿瘤的体积,但是同时在部分病人中也观察到了多方面的副作用.这些结果显示,VEGF也具有非血管新生方面的重要功能.因此,在研制基于拮抗VEGF作用的抗癌药物时,这些功能更不容忽视.研究表明,在成体的小肠、胰岛、甲状腺、肾脏和肝脏等器官组织中,VEGF都发挥着十分重要的作用,如果VEGF水平降低,这些器官组织的毛细血管网状结构将部分退化.VEGF还可以促进骨髓形成、组织修复与再生、促进卵巢囊泡成熟,并且参与血栓、炎症反应和缺氧缺血的病理过程.本文主要对VEGF在血管新生之外的功能及其分子机制进行了简要探讨.     

Signaling via vascular endothelial growth factor receptors

Angiogenesis, or development of blood vessels from preexisting vasculature, has important functions under both normal and pathophysiological conditions. Vascular endothelial growth factor receptors 1-3, also known as flt-1, KDR, and flt-4, are endothelial cell-specific receptor tyrosine kinases which serve as key mediators of the angiogenic responses. The review focuses on the signaling pathways that are initiated from these receptors and the recently identified VEGF coreceptor neuroplilin-1.     

Transforming growth factor-β1 stimulates macrophage urokinase expression and release of matrix-bound basic fibroblast growth factor

Macrophage expression of urokinase-type plasminogen activator (uPA) appears to play a role in their release of matrix-bound basic fibroblast growth factor (bFGF) and transforming growth factor-β (TGF-β). In experiments reported here, we have examined the potential regulatory effects of bFGF and TGF-β1 on macrophage uPA expression. TGF-β1 stimulated in a dose- and time-dependent manner the expression of secreted membrane and intracellular uPA activities by a macrophage cell line (RAW264.7). When examined at similar concentrations, bFGF had little effect, and interleukin-1α, tumor necrosis factor-α, and monocyte colony stimulating factor had no effect on macrophage uPA expression. Exposure of macrophages to TGF-β1 led to a rapid and sustained increase in the steady-state levels of uPA mRNA that was independent of de novo protein synthesis and was completely inhibited by actinomycin D. However, the TGF-β1-induced increase in uPA mRNA was largely unaffected by subsequent incubation of cells with actinomycin D. The protein kinase C inhibitior H7 markedly reduced the ability of TGF-β1 to stimulate expression of uPA activity. Likewise, okadaic acid and microcystin, inhibitors of serine/threonine phosphatases, potentiated the ability of TGF-β1 to upregulate macrophage uPA expression. TGF-β1 primed cells converted nearly all added plasminogen to plasmin and expressed sixfold more membrane-bound plasmin than control cells. Preincubation of TGF-β1 with either serum or methylamine-modified α2-macroglobulin did not affect its ability to induce macrophage uPA expression. When control and TGF-β1-primed macrophages were cultured on matrices containing bound¹²⁵I-bFGF, their release of ¹²⁵I-bFGF was increased five and tenfold, respectively, in the presence of plasminogen. The ability of TGF-β to induce macrophage uPA expression and the plasmin-dependent release of matrix-bound bFGF may provide an indirect mechanism by which TGF-β stimulates angiogenesis. © 1993 Wiley-Liss, Inc.     

血管内皮生长因子的脑保护及其机制研究进展

血管内皮生长因子(vascular endothelial growth factor,VEGF)是一种重要的血管发育调节因子,最早发现于肿瘤细胞。上世纪90年代,人们发现VEGF在神经细胞上也有广泛表达,并具有神经细胞保护作用。此外,VEGF显著促进成年哺乳动物结构性神经元再生区(constitutive neurogenic regions)和非神经元再生区(non-neurogenic regions)的神经元再生/更新(neurogenesis/regenera-tion),显示了VEGF在神经损伤性及退行性疾病治疗中的潜在意义。本文着重讨论VEGF在脑缺血损伤中的神经保护(neuroprotection)和神经修复(neural repair)及其细胞和分子机制研究进展。