血管内皮生长因子与肿瘤

血管内皮生长因子是新近确定的一种具有旁分泌机制的生长因子,能特异作用于血管内皮细胞,促进其增殖及新生血管的形成,同时还有增加血管通透性的作用．由于其生物学活性与实体瘤的生长密切相关,因此对它的研究倍受关注,进展非常迅速．     

G Protein α Subunit 14 Mediates Fibroblast Growth Factor 2-Induced Cellular Responses in Human Endothelial Cells

During pregnancy, a tremendous increase in fetoplacental angiogenesis is associated with elevated blood flow. Aberrant fetoplacental vascular function may lead to pregnancy complications including pre-eclampsia. Fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor A (VEGFA) are crucial regulators of fetoplacental endothelial function. G protein α subunit 14 (GNA14), a member of Gαq/11 subfamily is involved in mediating hypertensive diseases and tumor vascularization. However, little is known about roles of GNA14 in mediating the FGF2- and VEGFA-induced fetoplacental endothelial function. Using human umbilical vein endothelial cells (HUVECs) cultured under physiological chronic low oxygen (3% O₂) as a cell model, we show that transfecting cells with adenovirus carrying GNA14 complementary DNA (cDNA; Ad-GNA14) increases (p < 0.05) protein expression of GNA14. GNA14 overexpression blocks (p < 0.05) FGF2-stimulated endothelial migration, whereas it enhances (p < 0.05) endothelial monolayer integrity (maximum increase of ~35% over the control at 24 hr) in response to FGF2. In contrast, GNA14 overexpression does not significantly alter VEGFA-stimulated cell migration, VEGFA-weakened cell monolayer integrity, and intracellular Ca⁺⁺ mobilization in response to adenosine triphosphate (ATP), FGF2, and VEGFA. GNA14 overexpression does not alter either FGF2- or VEGFA-induced phosphorylation of ERK1/2. However, GNA14 overexpression time-dependently elevates (p < 0.05) phosphorylation of phospholipase C-β3 (PLCβ3) at S1105 in response to FGF2, but not VEGFA. These data suggest that GNA14 distinctively mediates fetoplacental endothelial cell migration and permeability in response to FGF2 and VEGFA, possibly in part by altering activation of PLCβ3 under physiological chronic low oxygen.     

Cloning and expression of human lectin-like oxidized low density lipoprotein receptor-1 in<Emphasis Type="Italic"> Pichia pastoris</Emphasis>

Lectin-like oxidatively-modified LDL receptor-1 (LOX-1) is a major receptor for oxidized low-density lipoprotein (oxLDL) in aortic endothelial cells. Human LOX-1 (hLOX-1) gene (cDNA) was cloned from the monocytic leukemic cell line THP-1 and expressed in Pichia pastoris. The recombinant protein (rhLOX-1) was purified by his-tag affinity chromatography. Preliminary identification was performed by Western blot analysis and a ligand-receptor binding assay showed that the protein had specific oxLDL-binding activity.Revisions requested 21 September 2004; Revisions received 10 November 2004     

Production of active pigment epithelium-derived factor in<Emphasis Type="Italic">E. coli</Emphasis>

Human pigment epithelium-derived factor (PEDF), a neurotrophic factor, is the most potent natural inhibitor of angiogenesis. To produce the active PEDF, the gene coding for the human PEDF protein was expressed in E. coli. The rPEDF protein was expressed at 457 mg l^–1 as a soluble protein. The yield of purified GST fusion protein was 14 mg ll^–1. Purified rPEDF inhibited tube formation in endothelial cells.Revisions requested 30 November 2004; Revisions received 25 January 2005     

A Simple and Green Procedure for the Microbial Effective Synthesis of 1-phenylethyl Alcohol in Both Enantiomeric Forms

Both R- and S-phenylethyl alcohol of high enantiomeric purity (98%) and with a satisfactory yield (40–80%) were obtained by bioreduction of acetophenone, catalyzed by whole cells of baker’s yeast. Revisions requested 29 November 2005; Revisions received 9 January 2006     

TBHQ激活自噬保护血管内皮细胞脂毒性作用的研究

目的:改善脂毒性引起的血管内皮细胞损伤在心血管疾病防治中发挥重要作用。本研究旨在探讨叔丁基对苯二酚(tert-butylhydroquinone,TBHQ)对脂毒性引起的血管内皮细胞损伤的保护作用。方法:以人脐静脉血管内皮细胞系EA.hy926为研究对象,给予不同浓度的饱和游离脂肪酸及TBHQ进行干预,检测细胞的凋亡情况。采用westernblotting及信号通路阻断技术对TBHQ的作用机制进行研究。结果:给与血管内皮细胞饱和游离脂肪酸进行干预可显著增加细胞死亡(P0.05)。TBHQ显著抑制饱和游离脂肪酸诱导的细胞死亡(P0.05)。激活自噬可显著抑制饱和游离脂肪酸引发的血管内皮细胞脂毒性(P0.05)。此外,TBHQ可显著激活血管内皮细胞的自噬(P0.05),采用自噬抑制剂可阻断TBHQ对脂毒性的保护作用(P0.05)。结论:TBHQ通过激活自噬有效地抑制饱和游离脂肪酸诱导的血管内皮细胞损伤,对于改善或防治高脂血症引起的血管损伤可能具有重要的现实意义。     

Endothelial activation markers and their key regulators after restrictive bariatric surgery

Objective: Increased plasma levels of endothelial activation markers in obese subjects reflect the positive association between cardiovascular diseases and obesity. The pro‐inflammatory state associated with obesity is thought to play a major role in endothelial cell activation in severely obese individuals. Previous studies demonstrated that long‐term weight loss after bariatric surgery is accompanied by a decreased proinflammatory state. However, little is known about the long‐term effects of bariatric surgery on endothelial cell activation. Research Methods and Procedures: Plasma levels of soluble intercellular adhesion molecule‐1 (sICAM‐1), soluble endothelial selectin (sE‐selectin), and soluble vascular cell adhesion molecule‐1 (sVCAM‐1), all markers of endothelial cell activation, and of their regulators adiponectin and resistin were measured at different time‐points postoperatively in 26 consecutive patients who underwent restrictive surgery, with a follow‐up of 2 years. Results: During the first 6 months after bariatric surgery, sE‐selectin levels decreased. Despite substantial weight loss, sICAM‐1 and sVCAM‐1 plasma levels did not decrease significantly. After 24 months, sICAM‐1 levels were significantly decreased, whereas sE‐selectin levels were further decreased. However, sVCAM‐1 levels remained elevated. Adiponectin levels did not change significantly during the first 6 months after bariatric surgery, whereas resistin levels increased. After 24 months, adiponectin levels were similar to normal‐weight controls, but resistin levels remained high. Discussion: Reductions in plasma levels of different markers of endothelial activation after bariatric surgery show different temporal patterns, suggesting that distinct mechanisms are involved in their regulation. Although not all endothelial activation markers normalize after bariatric surgery, our findings suggest that bariatric surgery can reduce endothelial activation in the long term.     

Increased Monocytic Adhesion by Senescence in Human Umbilical Vein Endothelial Cells

We investigated whether replicative senescence of endothelial cells contributed to the pathogenesis of atherosclerosis in human umbilical vein endothelial cells (HUVECs). HUVECs at a population-doubling level of 30 (PDL30) divided much more slowly than those at PDL9. The percentage of SA-β-Gal-positive cells and the mRNA expression levels of PAI-1 and p21 at PDL30 were significantly higher than those at PDL9. The changes induced by aging were evaluated according to the mRNA expression level of genes related to the endothelial cell function. The expression level of many adhesion molecules promoting monocytic adhesion was significantly increased, and monocytic adhesion on HUVECs was found to be significantly promoted by aging. Monocytic adhesion is an essential early event in the development of atherosclerosis, and our results suggest that replicative senescence of the vascular endothelial cells induced increased expression of adhesion molecules. The consequent increase in monocytic adhesion may then promote the pathogenesis of atherosclerosis.     

Biotransformation of racemic diisophorone by <Emphasis Type="Italic">Cephalosporium aphidicola</Emphasis> and <Emphasis Type="Italic">Neurospora crassa</Emphasis>

Racemic diisophorone (500 mg) was converted by Cephalosporium aphidicola and Neurospora crassa over 10 days at 25 °C to 8β-hydroxydiisophorone in yields of 10% (52 mg) and 20% (103 mg), respectively. The structure was established by IR, specific rotation, mass spectral, 1D and 2D-NMR studies.Revisions requested 2 March 2005 and 21 April 2005; Revisions received 8 April 2005 and 10 May 2005     

血管内皮细胞衰老与血管疾病

细胞衰老是指细胞在各种应激条件下出现周期阻滞,不可逆地丧失增殖能力,其形态、基因表达和功能都发生特定变化的过程。研究表明,血管内皮细胞衰老可以通过削弱血管功能,促进衰老相关血管疾病的发生发展。然而,有关内皮细胞衰老的发生机制以及内皮细胞衰老影响血管功能及衰老相关血管疾病的潜在机制尚待挖掘。本文从血管内皮细胞衰老相关的信号通路,以及血管内皮细胞衰老与血管功能和血管相关疾病（动脉粥样硬化、高血压和糖尿病血管并发症）的最新研究进展进行综述,为进一步认识血管疾病的发病机制,延缓血管衰老提供新的思路。     

Pre-germinated conidia of Coniothyrium minitans enhances the foliar biological control of Sclerotinia sclerotiorum

The relatively slow germination rate of Coniothyrium minitans limits its control efficiency against Sclerotinia sclerotiorum. Pre-germinated conidia of C. minitans enhanced its efficiency significantly: in foliar experiments with oilseed rape, hyphal extension of S. sclerotiorum was inhibited by 68%, while formation of sclerotia was completely inhibited when pre-germinated conidia were applied.Revisions requested 27 July 2004; Revisions received 7 September     

Regulation of Vascular Endothelial Growth Factor Receptor-2 (Flk-1) Expression in Vascular Endothelial Cells

We have previously reported the existence of a synergistic interaction between vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in the induction of angiogenesisin vitro.Here we demonstrate that bFGF increases VEGF receptor-2 (VEGFR-2/Flk-1) expression: mRNA levels were increased by 4.5- to 8.0-fold and total protein by 2.0- to 3.5-fold, in bovine microvascular endothelial (BME), aortic endothelial (BAE), and transformed fetal aortic (GM7373) endothelial cells. VEGF itself did not affect VEGFR-2 expression, and neither bFGF nor VEGF altered expression of FGF receptor-1. We also show that synergism occurs at the level of proliferation when this is measured in a three-dimensional but not in a conventional two-dimensional assay. Differences in the level of VEGFR-2 expression were also observed when cells were grown on or within collagen gels under different conditions: mRNA levels were lowest under sparse conditions, increased 20- to 26-fold at confluence, and increased even further (57-fold) when cells were cultured in suspension in three-dimensional collagen gels. Finally, a synergistic increase was seen in the level of expression of urokinase and urokinase receptor mRNAs when cells were exposed to bFGF and VEGF for 4 days. These findings demonstrate that the level of VEGFR-2 expression can be modulated by environmental factors including cytokines and the geometry of the culture conditions and provide some insight into the mechanisms of synergism between bFGF and VEGF in the induction of angiogenesisin vitro.     

High level expression of kringle 5 fragment of plasminogen in <Emphasis Type="Italic">Pichia</Emphasis><Emphasis Type="Italic">pastoris</Emphasis>

Angiogensis can be blocked by inhibitors such as endostatin and angiostatin. The kringle 5 fragment of plasminogen also has a potent inhibitory effect on endothelial cell proliferation and leads to the inhibition of angiogenesis. It has promise in anti-angiogenic therapy due to its small size and potent inhibitory effect. Preparation of kringle 5 has been achieved through the proteolysis of native plasminogen and recombinant DNA technology. Bacterially expressed recombinant kringle 5 is mainly insoluble and expressed at low level. The refolding yield is also low. To produce recombinant human kringle 5 in a large quantity, we have genetically modified a strain of Pichia pastoris. On methanol induction, this strain expressed and secreted biologically active, recombinant kringle 5. The expression level of the engineered strain in culture reached more than 300mgl^-1. Purification was easily achieved by precipitation, hydrophobic and DEAE ion exchange chromatography. The recovery of recombinant kringle 5 was about 50% after purification. Yeast-expressed kringle 5 has a higher activity in anti-endothelial proliferation than bacterially expressed kringle 5.Revisions requested 9 November 2004; Revisions received 2 December 2004     

Troglitazone‐Induced Changes in Adiponectin Do Not Affect Endothelial Function in Diabetes

Objective: Adiponectin has been proposed to be related to endothelial function. We have examined the relationship between the increase in adiponectin levels that is associated with troglitazone treatment and endothelium‐dependent vasodilation in type 2 diabetic patients. Research Methods and Procedures: Seventy‐two patients participated in this randomized, placebo‐controlled, double‐blinded study. High‐resolution ultrasound images were used to measure the flow‐mediated dilation (endothelium‐dependent) and nitroglycerin‐induced dilation (endothelium‐independent) of the brachial artery. Laser Doppler perfusion imaging was employed to measure the vascular reactivity in the forearm skin. Results: Troglitazone treatment resulted in an average 75% increase in the adiponectin levels, but no changes were observed in the endothelium‐dependent vasodilation, any other measurement of vascular reactivity, or any other markers of endothelial activation. Also, no changes were observed in the expression of the receptor for advanced glycation end‐products in skin biopsies taken from the forearm. Significant correlations were observed during troglitazone treatment between the changes in the adiponectin levels and the changes in fasting plasma glucose (r = ?0.29, p < 0.05), hemoglobin A_1c (r = ?0.30, p < 0.05), total cholesterol (r = 0.25, p < 0.05), and low‐density lipoprotein‐cholesterol (r = 0.34, p < 0.01). Discussion: The increase in adiponectin levels after troglitazone treatment is not associated with an improvement in the endothelium‐dependent vasodilation, indicating that adiponectin is not a major determinant of endothelial function. In addition, receptor for advanced glycation end‐products expression in the skin microcirculation is not affected by troglitazone treatment.     

Improvement of Bacillus thuringiensis δ-endotoxins Synthesis Yields Through Acquisition of Erythromycin Resistance

The acquisition of the erythromycin resistance by Bacillus thuringiensis kurstaki improved yields of δ-endotoxins in sporulating cells ranging from 134 to 215%. Resistance to erythromycin decreased the final spore count by at least 50%. Consequently, erythromycin resistance is an efficient tool for the improvement of bioinsecticides yields with a high ratio of δ-endotoxins to spores. Revisions requested 31 October 2005; Revisions received 28 November 2005     

Endothelial primary cilia inhibit atherosclerosis

Primary cilia are microtubule‐based structures present on most mammalian cells that are important for intercellular signaling. Cilia are present on a subset of endothelial cells where they project into the vessel lumen and are implicated as mechanical sensors of blood flow. To test the in vivo role of endothelial cilia, we conditionally deleted Ift88, a gene required for ciliogenesis, in endothelial cells of mice. We found that endothelial primary cilia were dispensable for mammalian vascular development. Cilia were not uniformly distributed in the mouse aorta, but were enriched at vascular branch points and sites of high curvature. These same sites are predisposed to the development of atherosclerotic plaques, prompting us to investigate whether cilia participate in atherosclerosis. Removing endothelial cilia increased atherosclerosis in Apoe^?/? mice fed a high‐fat, high‐cholesterol diet, indicating that cilia protect against atherosclerosis. Removing endothelial cilia increased inflammatory gene expression and decreased eNOS activity, indicating that endothelial cilia inhibit pro‐atherosclerotic signaling in the aorta.     

Perinuclear translocation of hsp 27 in shear stress exposed human endothelial cells

Expression of hsp 27 in human umbilical vein endothelial cells exposed to a shear stress was investigated. Using immunostaining, it was concluded that shear stress results in perinuclear translocation of hsp 27. Polymerization of actin microfilaments plays an important role in this process.Revisions requested 11 October 2004; Revisions received 2 February 2005     

雷帕霉素对内皮细胞迁移和血管内皮生长因子表达的影响

目的:研究不同浓度的雷帕霉素对体外培养的人血管内皮细胞(VE)迁移及血管内皮生长因子(VEGF)表达的影响。方法:用含10%胎牛血清的细胞培养基(DMEM)培养正常VE细胞,用10nM,50nM,100nM和200nM的雷帕霉素孵育VE细胞24 h,Western bloting测定雷帕霉素对VE中mTOR和VEGF表达的影响,Transwell迁移模型观察不同浓度的雷帕霉素对内皮细胞迁移影响。结果:①雷帕霉素可显著抑制VE的迁移,除了在100nM之外,基本呈浓度依赖性的。100nM雷帕霉素对VE迁移的抑制作用显著减弱(P<0.01)。②雷帕霉素对mTOR和VEGF165的表达呈浓度依赖性的抑制;而VEGF121的表达则是先升高后降低,在100nM雷帕霉素时表达最高,远远高于该浓度雷帕霉素时VEGF165的表达,可以解释100nM雷帕霉素时VE迁移抑制显著减轻的现象。结论:雷帕霉素抑制了VEGF165的表达,并且其对VE迁移抑制的效应主要由VEGF165表达减少所介导。VEGF121的表达在一定雷帕霉素浓度范围内可显著上调,从而显著改善了雷帕霉素诱导的VEGF165表达减少所致的内皮细胞迁移抑制。     

Actin cytoskeletal isoforms in human endothelial cellsin vitro: Alteration with cell passage

Summary The microfilamentous actin component of the cytoskeleton is crucial to endothelial angiogenesis and vascular permeability. Differences in actin cytoskeletal profiles in cultured human endothelial cells were explored: when first isolated, both primary human umbilical vein endothelial cells (HUVEC) and primary human placental microvascular endothelial cells (HPMEC) expressed F-actin, but notβ-actin orα-smooth muscle actin. A similar endothelial actin profile was observed in cryo-sections of freshly delivered term umbilical cord and placenta. In subsequent cell culture, although the actin cytoskeleton of HUVEC remained unchanged, the actin profiles of HPMEC altered after the second passage with the induction ofα-smooth muscle actin expression, which was intercellularly heterogeneous and increased to 20% at P4. This behavior occurred in HPMEC monolayers cultured on a variety of extracellular matrices. Comparisons with a spontaneously immortalized human microvascular cell-line, HGTEN 21, revealed that inprolonged passage, bothα-smooth muscle actin andβ-actin were expressed, whereas HPMEC at P4 showed a lower level ofβ-actin expression. Therefore, in comparison with large vessels, microvascular cells are more likely to dedifferentiate. This may reflect the ability of microvascular cells to remodel according to changing requirement for new vessel formation. In conclusion, passage of human microvascular endothelial cells, but not of larger vessel endothelial cells, alters the expression of actin isoforms. This may be important in relation to comparisons ofin vitro andin vivo vascular permeability; higher passage microvascular endothelial cells should thus be used with caution in such studies.     

Textile dye decolorization using cyanobacteria

Cyanobacterial cultures isolated from sites polluted by industrial textile effluents were screened for their ability to decolorize cyclic azo dyes. Gloeocapsa pleurocapsoides and Phormidium ceylanicum decolorized Acid Red 97 and FF Sky Blue dyes by more than 80% after 26 days. Chroococcus minutus was the only culture which decolorized Amido Black 10B (55%). Chlorophyll a synthesis in all cultures was strongly inhibited by the dyes. Visible spectroscopy and TLC confirmed that color removal was due to degradation of the dyes.Revisions requested 10 November 2004/30 November 2004; Revisions received 16 November 2004/ 7 January 2005