Improved angiogenic potency by implantation of ex vivo hypoxia prestimulated bone marrow cells in rats

Therapeutic angiogenesis can be induced by local implantation of bone marrow cells. We tried to enhance the angiogenic potential of this treatment by ex vivo hypoxia stimulation of bone marrow cells before implantation. Bone marrow cells were collected and cultured at 33 degrees C under 2% O(2)-5% CO(2)-90% N(2) (hypoxia) or 95% air-5% CO(2) (normoxia). Cells were also injected into the ischemic hindlimb of rats after 24 h of culture. Hypoxia culture increased the mRNA expression of vascular endothelial growth factor (VEGF), vascular endothelial (VE)-cadherin, and fetal liver kinase-1 (Flk-1) from 2.5- to fivefold in bone marrow cells. The levels of VEGF protein in the ischemic hindlimb were significantly higher 1 and 3 days after implantation with hypoxia-cultured cells than with normoxia-cultured or noncultured cells. The microvessel density and blood flow rate in the ischemic hindlimbs were also significantly (P < 0.001) higher 2 wk after implantation with hypoxia-cultured cells (89.7 +/- 5.5%) than with normoxia-cultured cells (67.0 +/- 9.6%) or noncultured cells (70.4 +/- 7.7%). Ex vivo hypoxia stimulation increased the VEGF mRNA expression and endothelial differentiation of bone marrow cells, which together contributed to improved therapeutic angiogenesis in the ischemic hindlimb after implantation.     

Production and secretion of adrenomedullin in cultured human alveolar macrophages.

To explore the role of adrenomedullin (ADM) in macrophages, we investigated the secretion of ADM by alveolar macrophages. Human alveolar macrophages obtained from bronchoalveolar lavage were cultured for 24 h. Northern blot analysis revealed ADM mRNA expression in alveolar macrophages. The levels of immunoreactive ADM in the media were 0.89+/-0.12 fmol/10(5) cells/24 h (n = 10). Reverse-phase high-performance liquid chromatography of the extract of culture media showed one major peak eluting in the position of the human ADM standard. The present study shows that alveolar macrophages produce and secrete ADM.     

Acute hypoxia alters eicosanoid production of perfused pulmonary artery endothelial cells in culture.

Hypoxia alters vascular tone which regulates regional blood flow in the pulmonary circulation. Endothelial derived eicosanoids alter vascular tone and blood flow and have been implicated as modulators of hypoxic pulmonary vasoconstriction. Eicosanoid production was measured in cultured bovine pulmonary endothelial cells during constant flow and pressure perfusion at two oxygen tensions (hypoxia: 4% O2, 5% CO2, 91% N2; normoxia: 21% O2, 5% CO2, 74% N2). Endothelial cells were grown to confluence on microcarrier beads. Cell cartridges (N = 8) containing 2 ml of microcarrier beads (congruent to 5 x 10(6) cells) were constantly perfused (3 ml/min) with Krebs' solutions (pH 7.4, T 37 degrees C) equilibrated with each gas mixture. After a ten minute equilibration period, lipids were extracted (C18 Sep Pak) from twenty minute aliquots of perfusate over three hours (nine aliquots per cartridge). Eicosanoids (6-keto PGF1 alpha; TXB2; and total leukotriene [LT - LTC4, LTD4, LTE4, LTF4]) were assayed by radioimmunoassay. Eicosanoid production did not vary over time. 6-keto PGF1 alpha production was increased during hypoxia (normoxia 291 +/- 27 vs hypoxia 395 +/- 35 ng/min/gm protein; p less than 0.01). Thromboxane production (normoxia 19 +/- 2 vs hypoxia 20 +/- 2 ng/min/gm protein) and total leukotriene production (normoxia 363 +/- 35 vs hypoxia 329 +/- 29 ng/min/gm protein) did not change with hypoxia. These data demonstrated that oxygen increased endothelial prostacyclin production but did not effect thromboxane or leukotriene production.     

线粒体ATP敏感钾通道对大鼠肺动脉平滑肌细胞低氧诱导因子-1α表达及细胞增殖的作用

本文旨在探讨线粒体ATP敏感钾（mitochondrial ATP-sensitive K＋,MitoKATP）通道对大鼠肺动脉平滑肌细胞低氧诱导因子-1α（hypoxia inducible factor-1α,HIF-1α）表达和细胞增殖的影响。原代培养大鼠肺动脉平滑肌细胞,分为常氧对照组、常氧＋diazoxide（MitoKATP通道的选择性开放剂）组、常氧＋5-hydroxydecanoate（5-HD,MitoKATP通道的选择性阻断剂）组、低氧对照组、低氧＋diazoxide组、低氧＋5-HD组,共6组,分别应用罗丹明123荧光技术检测各组大鼠肺动脉平滑肌细胞的线粒体膜电位,免疫组化检测HIF-1α的表达及酶联免疫检测仪检测细胞增殖的变化。结果显示,常氧＋ diazoxide组与常氧对照组比较,罗丹明123荧光、HIF-1α表达及细胞增殖明显增强（P〈0．05）;低氧＋diazoxide组与低氧对照组比较,罗丹明123荧光、HIF-1α表达及细胞增殖明显增强（P〈0.05）：常氧＋5-HD组与常氧对照组比较,罗丹明123荧光、HIF-1α表达、细胞增殖没有明显变化（P〉0．05）;但低氧＋5-HD组与低氧对照组比较,罗丹明123荧光明显减弱、HIF-1α表达及细胞增殖有所减弱（P〈0．05）。结果提示：MitoKATP通道的开放能引起大鼠肺动脉平滑肌细胞线粒体膜去极化,并可以促进HIF-1α的表达及细胞增殖。     

Selective recruitment of endothelial progenitor cells to ischemic tissues with increased neovascularization

Tissue ischemia remains a common problem in plastic surgery and one for which proangiogenic approaches have been investigated. Given the recent discovery of circulating endothelial stem or progenitor cells that are able to form new blood vessels, the authors sought to determine whether these cells might selectively traffic to regions of tissue ischemia and induce neovascularization. Endothelial progenitor cells were isolated from the peripheral blood of healthy human volunteers and expanded ex vivo for 7 days. Elevation of a cranially based random-pattern skin flap was performed in nude mice, after which they were injected with fluorescent-labeled endothelial progenitor cells (5 x 10(5); n = 15), fluorescent-labeled human microvascular endothelial cells (5 x 10(5); n = 15), or media alone (n = 15). Histologic examination demonstrated that endothelial progenitor cells were recruited to ischemic tissue and first appeared by postoperative day 3. Subsequently, endothelial progenitor cell numbers increased exponentially over time for the remainder of the study [0 cells/mm2 at day 0 (n = 3), 9.6 +/- 0.9 cells/mm2 at day 3 (n = 3), 24.6 +/- 1.5 cells/mm2 at day 7 (n = 3), and 196.3 +/- 9.6 cells/mm2 at day 14 (n = 9)]. At all time points, endothelial progenitor cells localized preferentially to ischemic tissue and healing wound edges, and were not observed in normal, uninjured tissues. Endothelial progenitor cell transplantation led to a statistically significant increase in vascular density in ischemic tissues by postoperative day 14 [28.7 +/- 1.2 in the endothelial progenitor cell group (n = 9) versus 18 +/- 1.1 in the control media group (n = 9) and 17.7 +/- 1.0 in the human microvascular endothelial cell group (n = 9; p < 0.01)]. Endothelial progenitor cell transplantation also showed trends toward increased flap survival [171.2 +/- 18 mm2 in the endothelial progenitor cell group (n = 12) versus 134.2 +/- 10 mm2 in the media group (n = 12) and 145.0 +/- 13 mm2 in the human microvascular endothelial cell group (n = 12)], but this did not reach statistical significance. These findings indicate that local tissue ischemia is a potent stimulus for the recruitment of circulating endothelial progenitor cells. Systemic delivery of endothelial progenitor cells increased neovascularization and suggests that autologous endothelial progenitor cell transplantation may have a role in the salvage of ischemic tissue.     

低氧对肺内小动脉平滑肌细胞血管紧张素转化酶活性和基因表达的影响

实验采用分离培养兔肺内小动脉平滑细胞,观察低氧对ＰＡＳＭＣ的血管紧张素转化酶活性和基因表达的影响。     

Increased expression of CXCR4 and integrin αM in hypoxia‐preconditioned cells contributes to improved cell retention and angiogenic potency

Cell‐based angiogenesis is a promising method for the treatment of ischemic diseases, but the poor retention of implanted cells in targeted tissues is a major drawback. We tested whether hypoxic preconditioning increased retention and angiogenic potency of implanted cells in ischemic tissue. Hypoxic preconditioning of mouse peripheral blood mononuclear cells (PBMNCs) was done with 24 h of culture under 2% O₂. Normoxia‐cultured PBMNCs were used as a control. Hypoxic preconditioning increased the adhesion capacity of the PBMNCs. Moreover, the expression of integrin αM and CXCR4 was significantly higher in the hypoxia‐preconditioned PBMNCs than in the normoxia‐cultured PBMNCs. Interestingly, the expression of intercellular adhesion molecule‐1 (ICAM‐1), a ligand of integrin αM, and stromal cell‐derived factor‐1 (SDF‐1), a chemokine for CXCR4, were remarkably increased in the ischemic hindlimbs. The retention of the hypoxia‐preconditioned PBMNCs was significantly higher than that of the normoxia‐cultured PBMNCs, 3 days after their intramuscular implantation into ischemic hindlimbs. We also noted better blood flow in the ischemic hindlimbs implanted with the hypoxia‐preconditioned PBMNCs than in those implanted with the normoxia‐cultured PBMNCs, 14 days after treatment. Furthermore, antibody neutralization of integrin αM and CXCR4 abolished completely the increased cell retention and angiogenic potency of the hypoxia‐preconditioned PBMNCs after implantation into the ischemic hindlimbs. These results indicate that hypoxic preconditioning of implanted cells is a feasible method of enhancing therapeutic angiogenesis by increasing their retention. J. Cell. Physiol. 220: 508–514, 2009. © 2009 Wiley‐Liss, Inc.     

低氧促进神经干细胞向多巴胺能神经元分化

神经干细胞（neural stem cells,NSCs）作为具有多向分化潜能的神经前体细胞,被广泛应用于细胞移植等研究,而低氧不但调节干细胞的体外增殖,在干细胞分化中也具有重要的作用。本文着重探讨了低氧对NSCs分化的调节作用。采用Wistar孕大鼠（E13．5d）,分离胚胎中脑NSCs,加入无血清DMEM／F12培养液（含20ng／mL EGF、20ng／mL bFGF、1％ N2和B27）,3～5d后传代,细胞培养至第三代进行诱导分化,分别在低氧（3％O2）和常氧（20％O2）条件下诱导分化3d,然后在常氧条件下分化成熟5～7d（DMEM／F12含1％FBS、N2和B27）后进行检测。Nestin、NeuN以及TH免疫组织化学鉴定NSCs;流式细胞术分析测定NSCs向TH阳性神经元方向的分化;高效液相色谱测定细胞培养上清液中多巴胺（dopamine,DA）含量。结果显示,分离培养的NSCs均为nestin阳性细胞;低氧可明显促进NSCs向神经元方向的分化;TH阳性神经元比例在常氧和低氧组分别为（10．25±1．03）％和（19．88±1．44）％。NSCs诱导分化7d后,低氧组细胞培养上清液中DA浓度明显增加,约为常氧组的2倍（P〈0．05,n=8）。上述结果表明,3％低氧可促进NSCs向神经元方向,特别是向DA能神经元方向分化。这为NSCs应用于临床治疗帕金森病提供了基础。     

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) inhibition of coronary vasculogenesis is mediated, in part, by reduced responsiveness to endogenous angiogenic stimuli, including vascular endothelial growth factor A (VEGF-A)

BACKGROUND: 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exposure prior to chick embryo incubation (GD 0) induces dilated cardiomyopathy, and reduces myocardial hypoxia, vascular endothelial growth factor A (VEGF-A) expression, and coronary vascularization. We investigated whether reduced coronary vascularization 1) occurs in the absence of changes in cardiac morphology and 2) is associated with altered secretion of VEGF-A and/or an antivasculogenic factor. METHODS: Chicken eggs were treated with control (corn oil) or TCDD (0.075-0.3 pmol of TCDD/gm) on GD 5. In vivo cardiac morphology and artery number were determined on GD 10, while in vitro vascular outgrowth and VEGF-A secretion were determined from cardiac explants on GD 6. Effects of recombinant VEGF-A (rcVEGF-A), soluble flt-1 (sFlt-1) receptor plus rcVEGF-A, and control conditioned media were assessed in TCDD explants, while effects of TCDD-conditioned media was assessed in control explants. RESULTS: TCDD reduced coronary artery number in vivo by 53 +/- 8% and induced a dose-related reduction in tube outgrowth in vitro, but had no effect on cardiac morphology. All TCDD doses reduced explant VEGF-A secretion equally (43 +/- 3%), compared to control. sFlt-1 blocked outgrowth in control cultures and blocked rcVEGF-A-mediated rescue of outgrowth in TCDD explants. Control conditioned media partially rescued outgrowth from TCDD explants, while conditioned media from TCDD explants had no effect on controls. CONCLUSIONS: TCDD inhibition of coronary vascularization can occur in the absence of changes in cardiac morphology and is associated with reduced VEGF-A secretion but not an antivasculogenic factor. Since control media only partly rescues TCDD's inhibitory effect, we suggest that TCDD-exposed endothelial cells are less responsive to vasculogenic stimuli.     

Nitric oxide/cGMP protects endothelial cells from hypoxia-mediated leakiness

Leakiness of the endothelial bed is attributed to the over-perfusion of the pulmonary bed, which leads to high altitude pulmonary edema (HAPE). Inhalation of nitric oxide has been successfully employed to treat HAPE patients. We hypothesize that nitric oxide intervenes in the permeability of the pulmonary macrovascular endothelial bed to rectify the leaky bed under hypoxia. Our present work explores the underlying mechanism of 'hypoxia-mediated' endothelial malfunction by using human umbilical cord-derived immortalized endothelial cells, ECV-304, and bovine pulmonary artery primary endothelial cells. The leakiness of the endothelial monolayer was increased by two-fold under hypoxia in comparison to cells under normoxia, while optical tweezers-based tethering assays reported a higher membrane tension of endothelial cells under hypoxia. Phalloidin staining demonstrated depolymerization of F-actin stress fibers and highly polarized F-actin patterns in endothelial cells under hypoxia. Nitric oxide, 8-Br-cGMP and sildenafil citrate (phosphodiesterase type 5 inhibitor) led to recovery from hypoxia-induced leakiness of the endothelial monolayers. Results of the present study also suggest that 'hypoxia-induced' cytoskeletal rearrangements and membrane leakiness are associated with the low nitric oxide availability under hypoxia. We conclude that nitric oxide-based recovery of hypoxia-induced leakiness of endothelial cells is a cyclic guanosine monophosphate (cGMP)-dependent phenomenon.     

雷帕霉素对低氧下HL-60细胞的低氧调控信号分子表达的影响

摘要目的：通过小分子化合物氯（CoCt2）模拟的低氧环境,分析低氧下及其雷帕霉素（RPM）作用下人急性髓细胞白血病细胞HL．60的低氧调控信号分子表达的变化;方法：常规方法复苏、传代、培养HL-60细胞,培养细胞进入对数生长期后用于实验。低氧模拟组、低氧雷帕霉素处理组、常氧雷帕霉素处理组分别用含2001xmol／LCoCl2、2001xmol／LCOCl2／20nmol／LRPM、20nmol／LRPM的1640培养基处理生长状态良好的细胞,对照组细胞用1640培养基培养,各组置培养箱以37℃、5％CO2培养,并于处理后24h、48h、72h收集细胞用于检测;采用实时荧光定量PCR方法检测低氧诱导因子（HIF-1α）、内皮细胞生长因于（VEGF）、雷帕霉素靶蛋白（mTOR）及GAPDH在转录水平的表达;结果：①与各时段对照组相比,低氧模拟组HIF-1α表达随时间逐渐增加,72h明显上调;与常氧雷帕霉素处理组各时段比较,低氧雷帕霉素处理组HIF-1α表达早期（24h）相对下调,后期相对上调;②．与对照组比较,各处理组mTOR表达均下调,低氧雷帕霉素处理组在早期（24h）下调显著;与常氧雷帕霉素处理组比较,低氧雷帕霉素处理组mTOR各时段的表达均相对下调;③与对照组各时段相比,低氧模拟组VEGF的表达在早期显著上调,但后期呈下调;常氧雷帕霉素处理组各时段VEGF的表达下调,与其比较,低氧雷帕霉素处理组各时段均呈相对下调。结论：常氧和低氧下RPM作用HL-60细胞后VEGF、mTOR的mRNA均表达下调,RPM可在低氧环境下增强了这种下调表达作用。     

Adrenomedullin expression in a rat model of acute lung injury induced by hypoxia and LPS

Adrenomedullin (ADM) is upregulated independently by hypoxia and LPS, two key factors in the pathogenesis of acute lung injury (ALI). This study evaluates the expression of ADM in ALI using experimental models combining both stimuli: an in vivo model of rats treated with LPS and acute normobaric hypoxia (9% O2) and an in vitro model of rat lung cell lines cultured with LPS and exposed to hypoxia (1% O2). ADM expression was analyzed by in situ hybridization, Northern blot, Western blot, and RIA analyses. In the rat lung, combination of hypoxia and LPS treatments overcomes ADM induction occurring after each treatment alone. With in situ techniques, the synergistic effect of both stimuli mainly correlates with ADM expression in inflammatory cells within blood vessels and, to a lesser extent, to cells in the lung parenchyma and bronchiolar epithelial cells. In the in vitro model, hypoxia and hypoxia + LPS treatments caused a similar strong induction of ADM expression and secretion in epithelial and endothelial cell lines. In alveolar macrophages, however, LPS-induced ADM expression and secretion were further increased by the concomitant exposure to hypoxia, thus paralleling the in vivo response. In conclusion, ADM expression is highly induced in a variety of key lung cell types in this rat model of ALI by combination of hypoxia and LPS, suggesting an essential role for this mediator in this syndrome.     

缺氧对食管癌细胞增殖及凋亡的影响

目的:探讨缺氧对人食管癌细胞Eca109增殖及凋亡的影响及其作用机制,为食管癌的诊断和治疗提供新的思路和路径。方法:以终浓度为250μmol/L氯化钴模拟缺氧环境,将人食管癌细胞Eca109于常氧及缺氧条件下分别培养12 h、24 h、36 h、48 h,采用倒置相差显微镜观察细胞生长情况,MTT法检测细胞增殖情况,利用细胞活力分析仪NC-3000检测各组细胞凋亡情况。结果:相对于常氧对照组而言,缺氧后各组Eca109细胞增殖减弱,凋亡细胞比率明显升高,其中以缺氧24 h的凋亡细胞比率最高,缺氧36 h的凋亡细胞比率较缺氧24 h时有所回落。结论:缺氧可以抑制人食管癌细胞Eca109的增殖并诱导其凋亡。     

A versatile in vitro assay for investigating angiogenesis of the heart

Neovascularization in the heart is usually investigated with models of angiogenesis in vivo. Here we present a simple model that allows investigating heart angiogenesis in mice and rats in vitro. Small pieces of left ventricular myocardium were cultured in three-dimensional fibrin gels for 10 days. A single mouse heart allowed assessing 24 conditions, each tested in octuplicates. Rat recombinant VEGF164, human recombinant bFGF, and human recombinant PDGF-BB were used under normoxia (21% O2) and hypoxia (3% O2), and outgrowth of endothelial sprouts from heart pieces was quantified. In 4-week-old OF1 mice, endothelial sprouts formed spontaneously. In contrast, in 12-week-old adult mice, virtually no sprouts formed under normoxia. Under hypoxia, sprout formation increased substantially. Different growth factors induced formation of distinct patterns of sprouts and unorganized single cells. Sprouts were composed of endothelial cells with smooth muscle cells or pericytes interacting with them, as assessed by immunohistochemistry. Taken together, our model is suited for investigation of angiogenesis of the heart in vitro. It may allow performing extensive series of experiments in vitro including rapid screening of pharmacological compounds and assessment of mechanisms of heart angiogenesis in transgenic animals in an easy straightforward manner.     

Activation and damage of endothelial cells upon hypoxia/reoxygenation. Effect of extracellular pH

Disturbances of blood flow upon vascular occlusions and spasms result in hypoxia and acidosis, while its subsequent restoration leads to reoxygenation and pH normalization (re-alkalization) in ischemic sites of the vascular bed. The effect of hypoxia/reoxygenation on activation and stimulation of apoptosis in cultured human endothelial cells was studied. The cells were subjected to hypoxia (2% O₂, 5% CO₂, 93% N₂) for 24 h followed by reoxygenation (21% O₂, 5% CO₂, 74% N₂) for 5 h. Reoxygenation was carried out at different pH-6.4 (preservation of acidosis after hypoxia), 7.0, and 7.4 (partial and complete re-alkalization, respectively). Hypoxia only slightly (by ～30%) increased the cell adhesion molecule ICAM-1 content on the cell surface, whereas reoxygenation more than doubled its expression. The reoxygenation effect depended on the medium acidity, and ICAM-1 increase was more pronounced at pH 7.0 compared to that at pH 6.4 and 7.4. Neither hypoxia nor reoxygenation induced expression of two other cell adhesion molecules, VCAM and E-selectin. Incubation of cells under hypoxic conditions but not reoxygenation stimulated secretion of von Willebrand factor and increased its concentration in the culture medium by more than 4 times. The percentage of cells containing apoptosis marker, activated caspase-3, was increased by approximately 1.5 times upon hypoxia as well as hypoxia/reoxygenation. Maximal values were achieved when reoxygenation was performed at pH 7.0. These data show that hypoxia/reoxygenation stimulate pro-inflammatory activation (ICAM-1 expression) and apoptosis (caspase-3 activation) of endothelial cells, and the extracellular pH influences both processes.     

Basic Fibroblast Growth Factor Contributes to a Shift in the Angioregulatory Activity of Retinal Glial (Müller) Cells

Basic fibroblast growth factor (bFGF) is a pleiotropic cytokine with pro-angiogenic and neurotrophic effects. The angioregulatory role of this molecule may become especially significant in retinal neovascularization, which is a hallmark of a number of ischemic eye diseases. This study was undertaken to reveal expression characteristics of bFGF, produced by retinal glial (Müller) cells, and to determine conditions under which glial bFGF may stimulate the proliferation of retinal microvascular endothelial cells. Immunofluorescence labeling detected bFGF in Müller cells of the rat retina and in acutely isolated Müller cells with bFGF levels, which increased after ischemia-reperfusion in postischemic retinas. In patients with proliferative diabetic retinopathy or myopia, the immunoreactivity of bFGF co-localized to glial fibrillary acidic protein (GFAP)-positive cells in surgically excised retinal tissues. RT-PCR and ELISA analyses indicated that cultured Müller cells produce bFGF, which is elevated under hypoxia or oxidative stress, as well as under stimulation with various growth factors and cytokines, including pro-inflammatory factors. When retinal endothelial cells were cultured in the presence of media from hypoxia (0.2%)-conditioned Müller cells, a distinct picture of endothelial cell proliferation emerged. Media from 24-h cultured Müller cells inhibited proliferation, whereas 72-h conditioned media elicited a stimulatory effect. BFGF-neutralizing antibodies suppressed the enhanced endothelial cell proliferation to a similar extent as anti-VEGF antibodies. Furthermore, phosphorylation of extracellular signal-regulated kinases (ERK−1/−2) in retinal endothelial cells was increased when the cells were cultured in 72-h conditioned media, while neutralizing bFGF attenuated the activation of this signaling pathway. These data provide evidence that retinal (glial) Müller cells are major sources of bFGF in the ischemic retina. Müller cells under physiological conditions or transient hypoxia seem to provide an anti-angiogenic environment, but long-lasting hypoxia causes the release of bFGF, which might significantly co-stimulate neovascularization in the retina.     

Hypoxia/reoxygenation increases the permeability of endothelial cell monolayers: role of oxygen radicals

We assessed the effect of hypoxia/reoxygenation on 14C-albumin flux across endothelial monolayers. Cultured bovine pulmonary artery endothelial cells were grown to confluence on nitrocellulose filters (pore size 12 microns). The endothelialized filters were mounted in Ussing-type chambers which were filled with cell culture medium (M 199). Equimolar amounts (33 nM) of 14C-labeled and unlabeled albumin were added to the "hot" and "cold" chambers, respectively. The monolayers were then exposed to successive periods (90 min) of normoxia (pO2 145 mmHg), hypoxia (pO2 20 mmHg), and reoxygenation (pO2 145 mmHg). A gas bubbling system was used to control media pO2 and to ensure adequate mixing. Four aliquots of culture media were taken during each period in order to calculate the 14C-albumin permeability across the endothelialized filter. In some experiments, either the xanthine oxidase inhibitor, oxypurinol (10 microM), or superoxide dismutase (600 U/mL), was added to the media immediately prior to the experiments. As compared to the normoxic control period, albumin permeability was 1.5 times higher during hypoxia (p less than 0.01) and 2.3 times higher during reoxygenation (p less than 0.01). The reoxygenation-induced increase in albumin permeability was prevented by either oxypurinol or superoxide dismutase. These data indicate that xanthine oxidase-derived oxygen radicals contribute to the hypoxia/reoxygenation-induced endothelial cell dysfunction. The altered endothelial barrier function induced by hypoxia/reoxygenation is consistent with the microvascular dysfunction observed following reperfusion of ischemic tissues.     

Peroxisome proliferator-activated receptor-gamma agonists increase vascular endothelial growth factor expression in human vascular smooth muscle cells

Vascular endothelial growth factor (VEGF), expressed in a variety of mesenchymal cells including vascular smooth muscle cells (VSMC), is a potent mitogen for endothelial cells, and is used clinically applied for ischemic disease of peripheral vessels. To determine whether peroxisome proliferator-activated receptor gamma (PPARgamma) regulates VEGF production in VSMC, we examined VEGF secretion from VSMC treated with PPAR agonists. Troglitazone increased VEGF secretion in a time- and dose-dependent manner (261 +/- 35% with 25 mM of troglitazone for 24 h), and also increased levels of VEGF mRNA. VEGF secretion was also increased by other PPARgamma agonists, pioglitazone, LY171883, and 15d-PGJ2 (224 +/- 17.1%, 247 +/- 36.8% and 171 +/- 7.8%, respectively), but not the PPARgamma agonists bezafibrate and Wy14643 (85.2 +/- 1.5%, 94.6 +/- 3.2, respectively). Our findings suggest that thiazolidinediones might be useful for the therapeutic angiogenesis for ischemic artery disease.