Supercritical carbon dioxide generated vascular endothelial growth factor encapsulated poly(DL-lactic acid) scaffolds induce angiogenesis in vitro

The ability to deliver, over time, biologically active vascular endothelial growth factor-165 (VEGF) through tailored designed scaffolds offers tremendous therapeutic opportunities to tissue-engineered therapies. Porous biodegradable poly(DL-lactic) acid (PLA) scaffolds encapsulating VEGF have been generated using supercritical CO2 (scCO2) and the kinetic release and angiogenic activity of these scaffolds examined in vitro and in an ex vivo chick chorioallantoic membrane (CAM) angiogenesis model. After processing through scCO2, VEGF maintained its angiogenic activity as assessed by increased tubule formation of human umbilical vein endothelial cells (HUVEC) cultured on Matrigel (VEGF = 1937 +/- 205 microm; scCO2-VEGF = 2085 +/- 234 microm; control = 1237 +/- 179 microm). VEGF release kinetics from scCO2-VEGF incorporated PLA monolith scaffolds showed a cumulative release of VEGF (2837 +/- 761 rhog/ml) over a 21 day period in culture. In addition, VEGF encapsulated PLA scaffolds increased the blood vessel network in the CAM compared to controls; control, 24.8 +/- 9.6; VEGF/PLA, 44.1 +/- 12.1 (vessels/field). These studies demonstrate that the controlled release of growth factors encapsulated into three-dimensional PLA scaffolds can actively stimulate the rapid development of therapeutic neovascularisation to regenerate or engineer tissues.     

Expression of VEGF and angiopoietins-1 and -2 during ischemia-induced coronary angiogenesis

The mechanisms underlying coronary capillary growth in response to ischemia are undefined. We hypothesized that the expression of vascular endothelial growth factor (VEGF) and angiopoietin (Ang)/Tie-2 were involved in capillary growth as an adaptation to ischemia. To test this hypothesis we measured capillary density, and the expressions of VEGF, Ang-1, Ang-2, and the Tie-2 receptor and its phosphorylation state during repetitive episodes of myocardial ischemia in chronically instrumented canines. Repetitive episodes of ischemia were induced by multiple (once/hour; 8/day), brief (2 min) occlusions of the left anterior descending coronary artery for 1, 7, 14, or 21 days. A sham group received the same instrumentation as the experimental groups but not the occlusion protocol. Collateral blood flow (microspheres) progressively increased from 9 +/- 3 to 83 +/- 10 ml. min-1. 100 g-1 on day 21. Capillary density increased at day 7 from 2378 +/- 53 (sham) to 2962 +/- 60/mm2, but it decreased to 2594 +/- 39/mm2 at day 21. Both VEGF and Ang-2 expression in myocardial interstitial fluid (Western analyses) peaked at day 3 of the repetitive occlusions but waned thereafter. In contrast the expression of Ang-1 remained relatively constant at all times in the occlusion groups. In shams, the expression of VEGF and Ang-2 was low and constant at all times. Tie-2 phosphorylation myocardial decreased decreased at day 7 but increased at 21 days of occlusions (P < 0.05). Our results indicate that capillary density was augmented by myocardial ischemia, but after development of collaterals and restoration of flow to the ischemic zone, capillary density returned to control levels. The change in capillary density paralleled with VEGF and Ang-2 expression but was inversely related to Tie-2 phosphorylation. We speculate the coronary angiogenesis is a coordinated event involving the expression of both VEGF and Ang-2 and that therapeutic angiogenic strategies may ultimately require treatment with more than a single factor.     

Human three-dimensional fibroblast cultures express angiogenic activity

Human neonatal fibroblasts were cultured on a lactate-glycollate copolymer scaffold for 12-16 days to form a three-dimensional dermal equivalent tissue. The cellular content of vascular endothelial growth factor (VEGF) mRNA in these three-dimensional cultures was 22-fold greater than that observed in the same fibroblasts grown as monolayers. No induction was shown by hepatocyte growth factor (HGF) or angiopoietin 1 indicating that the effect was specific to VEGF. The predominant VEGF splice variant, detected by RT-PCR corresponded to the 121 amino acid form, with less of the 165 amino acid form. The cell-associated forms (189 and 206 amino acids) comprised less than 1% of the total VEGF mRNA. VEGF and HGF proteins, determined by ELISA, were secreted in physiologically significant amounts, 0.5-4 ng per 24 h/10(6) cells. Conditioned medium from the three-dimensional cultures stimulated proliferation of endothelial cells in a dose-dependent manner and induced cellular expression of integrin alpha(v)beta(3). Conditioned medium from the same dermal fibroblasts cultured in monolayer showed little angiogenic activity in any of these assays. Using the chorioallantoic membrane (CAM) angiogenesis assay, the cultures stimulated blood vessel production 2.8-fold over scaffold alone. VEGF-neutralizing antibody reduced the vessel development in the CAM to the level in the scaffold control. Anti-HGF antibody had no significant effect. In conclusion, three-dimensional cultures of dermal equivalent tissue express angiogenic activity to a greater extent than monolayer cultures, some of which can be assigned to VEGF.     

血管内皮细胞生长因子受体Flt—1胞外区基因的克隆及其在中国仓？…

朋原代培养的人脐静脉血管内皮细胞（ＨＵＶＥＣ）提取细胞总ＲＮＡ,采用逆转录ＰＣＲ（ＲＴ－ＰＣＲ）方法得到ＶＥＧＦ受体Ｆｌｔ－１胞外区前３个ＩｇＧ样区域ｃＤＮＡ片段（Ｆｌｔ－１ｎ３）。将获得的受体基因克隆到真核表达载体ｐｃＤ－ＮＡ３．１中,得到重组质粒ｐｃＤＮＡ３．１／Ｆｌｔ－１ｎ３,通过南体转染方法将其转入中国仓鼠卵巢细胞（ＣＨＯ）,用Ｇ４１８筛选得到稳定表达目的蛋白的细胞砍隆。经固相结合实验筛选     

The effects of indomethacin on angiogenic factors mRNA expression in renal cortex of healthy rats.

Indomethacin is a nonsteroidal anti-inflammatory drug used frequently to control chronic or temporary pain. In the kidney, indomethacin decreases medullary and cortical perfusion, resulting in hypoxia. Kidney hypoxia has many effects, including changes in gene expression, and is a strong stimulus for angiogenesis. Other angiogenic factors include vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF-2), transforming growth factor beta 1 (TGFbeta1), and platelet-derived growth factor (PDGF). Our goal was to examine the influence of indomethacin on mRNA expression of these factors and their selected receptors in the renal cortex of healthy rats. Groups of 8 healthy, male, six-week-old Wistar rats received either indomethacin (5 mg/kg/day) or placebo orally for three months. RNA from renal cortex biopsies was analyzed by real-time polymerase chain reaction to quantify the mRNA levels of each cytokine. We observed significantly higher mRNA levels for VEGF (1.73-fold), FGF-2 (5.6-fold) and TGFbeta receptor III (2.93-fold), PDGF receptor alpha (2.93-fold) and receptor beta (2.91-fold) in rats receiving indomethacin compared to rats given placebo (p < 0.05). Amounts of mRNA for TGFbeta1, PDGF, FGF receptors 1 and 2 and TGFbeta receptor I did not differ between analysed groups. Our data indicates that indomethacin may regulate the expression of potent angiogenic factors VEGF and FGF-2.     

Acute ethanol increases angiogenic growth factor gene expression in rat skeletal muscle.

Moderate ethanol consumption demonstrates a protective effect against cardiovascular disease and improves insulin sensitivity, possibly through angiogenesis. We investigated whether 1) ethanol would increase skeletal muscle growth factor gene expression and 2) the effects of ethanol on skeletal muscle growth factor gene expression were independent of exercise-induced growth factor gene expression. Female Wistar rats were used. Four groups (saline + rest; saline + exercise; 17 mmol/kg ethanol + rest; and 17 mmol/kg ethanol + exercise) were used to measure the growth factor response to acute exercise and ethanol administration. Vascular endothelial growth factor (VEGF), transforming growth factor-beta(1) (TGF-beta(1)), basic fibroblast growth factor (bFGF), Flt-1, and Flk-1 mRNA were analyzed from the left gastrocnemius by quantitative Northern blot. Ethanol increased VEGF, TGF-beta(1), bFGF, and Flt-1 mRNA at rest and after acute exercise. Ethanol increased resting Flk-1 mRNA. Ethanol increased bFGF mRNA independently of exercise. These findings suggest that 1) ethanol can increase skeletal muscle angiogenic growth factor gene expression and 2) the mechanisms responsible for the ethanol-induced increases in VEGF, TGF-beta(1), and Flt-1 mRNA appear to be different from those responsible for exercise-induced regulation. Therefore, these results provide evidence in adult rat tissue that the protective cardiovascular effects of moderate ethanol consumption may result in part through the increase of angiogenic growth factors.     

DNA chip analysis of comprehensive food function: inhibition of angiogenesis and telomerase activity with unsaturated vitamin E, tocotrienol

Inhibition of angiogenesis and telomerase activity with vitamin E compounds, especially for tocotrienol (T3), has been investigated. Nutrigenomic tools have been used for elucidating the bioactive mechanisms of T3. In the cell culture experiments, T3 reduced the vascular endothelial growth factor (VEGF)-stimulated tube formation by human umbilical vein endothelial cells (HUVEC). Among T3 isomers, delta-T3 appeared the highest activity. The T3 inhibited the new blood vessels formation on the growing chick embryo chorioallantoic membrane (CAM assay for an in vivo model of angiogenesis). In contrast, tocopherol did not. The findings suggested that the T3 has potential use for reducing angiogenic disorder. DNA chip analysis revealed that T3 specifically down-regulates the expression of VEGF receptor (VEGFR) in endothelial cells. It is well-known that VEGF regulates angiogenesis by binding to VEGFR. Therefore, T3 could block the intracellular signaling of VEGF via down-regulation of VEGFR, which resulted in the inhibition of angiogenesis. On the other hand, DNA chip analysis also revealed that T3 down-regulates the expression of protein kinase C (PKC) in the cultured HUVEC. Since PKC is involved with the control of telomerase activity, T3 has potential to act as anti-telomerase inhibitor via PKC inhibition. In this manner, DNA chip technology provides efficient access to genetic information regarding food function and its mechanism.     

Angiogenesis and diabetes: different responses to pro-angiogenic factors in the chorioallantoic membrane assay

Hyperglycemia induces defects in angiogenesis without alteration in the expression of major vascular growth factors in the chicken chorioallantoic membrane (CAM) model. A direct negative effect of hyperglycemia on angiogenesis may participate in failures of "therapeutic angiogenesis" trials. Here, we tested the hypothesis that the response to pro-angiogenic molecules such as angiotensin-converting enzyme (ACE), endothelin-1 (ET-1), and vascular endothelial growth factor-A (VEGF) is altered by hyperglycemia. Transfected (Chinese hamster ovary [CHO] or human embryonic kidney [HEK]) cells overexpressing ACE, ET-1, or VEGF were deposed onto the CAM of hyperglycemic or control embryos. The proangiogenic effect was evaluated 3 d later by angiography and histological analyses. Gene expression in response to these factors was assessed by in situ hybridization. Only VEGF overexpression evoked a proangiogenic response in the CAM from hyperglycemic embryos, upregulating the expression of endogenous VEGF, VEGF-R2, and Tie-2, all of them related to activation of endothelial cells. In conclusion, in a model where hyperglycemia does not alter the major vascular growth factor expression, the negative effect of diabetes on capillary density was overcome only by VEGF overexpression, whereas responses to other vasoactive peptides were practically abolished under hyperglycemic conditions.     

Increased expression of HIF-1alpha, nNOS, and VEGF in the cerebral cortex of anemic rats

This study tested the hypothesis that specific hypoxic molecules, including hypoxia-inducible factor-1alpha (HIF-1alpha), neuronal nitric oxide synthase (nNOS), and vascular endothelial growth factor (VEGF), are upregulated within the cerebral cortex of acutely anemic rats. Isoflurane-anesthetized rats underwent acute hemodilution by exchanging 50% of their blood volume with pentastarch. Following hemodilution, mean arterial pressure and arterial Pa(O(2)) values did not differ between control and anemic rats while the hemoglobin concentration decreased to 57 +/- 2 g/l. In anemic rats, cerebral cortical HIF-1alpha protein levels were increased, relative to controls (1.7 +/- 0.5-fold, P < 0.05). This increase was associated with an increase in mRNA levels for VEGF, erythropoietin, CXCR4, iNOS, and nNOS (P < 0.05 for all), but not endothelial NOS. Cerebral cortical nNOS and VEGF protein levels were increased in anemic rats, relative to controls (2.0 +/- 0.2- and 1.5 +/- 0.4-fold, respectively, P < 0.05 for both). Immunohistochemistry demonstrated increased HIF-1alpha and VEGF staining in perivascular regions of the anemic cerebral cortex and an increase in the number of nNOS-positive cerebral cortical cells (3.2 +/- 1.0-fold, P < 0.001). The nNOS-positive cells costained with the neuronal marker, Neu-N, but not with the astrocytic marker glial fibrillary acidic protein (GFAP). These nNOS-positive neurons frequently sent axonal projections toward cerebral blood vessels. Conversely, VEGF immunostaining colocalized with both neuronal (NeuN) and astrocytic markers (GFAP). In conclusion, acute normotensive, normoxemic hemodilution increased the levels of HIF-1alpha protein and mRNA for HIF-1-responsive molecules. nNOS and VEGF protein levels were also increased within the cerebral cortex of anemic rats at clinically relevant hemoglobin concentrations.     

Discovery of a novel control element within the 5'-untranslated region of the vascular endothelial growth factor. Regulation of expression using sense oligonucleotides

The regulation of vascular endothelial growth factor (VEGF), a potent stimulator of angiogenesis, is controlled primarily through the interactions of control elements located within the 5'- and 3'-untranslated regions, many of which are yet to be described. In this study we examined the 5'-untranslated region of human VEGF for control elements with the aim of regulating expression both in vitro and in vivo using oligonucleotide gene therapy. A potential control element was located, two sense oligonucleotides (S(1) and S(2)) were designed based on its sequence, and a third oligonucleotide (S(3)) was designed as a control and mapped to the 16 base pairs immediately upstream. Retinal cells cultured in the presence of S(1) and S(2) resulted in a 2-fold increase of VEGF protein and a 1.5-fold increase in mRNA 24 h post-transfection whereas S(3) had no significant effect (p > 0.05) compared with controls. Subsequent reporter gene studies confirmed the necessity of this element for up-regulation by S(1). Further in vivo studies showed that S(1) and S(2) mediated an increase in VEGF protein in a rodent ocular model that resulted in angiogenesis. In addition to providing insight into the regulation of the vascular endothelial growth factor, the use of these oligonucleotides to stimulate vascular growth may prove useful for the treatment of ischemic tissues such as those found in the heart following infarct.     

人骨肉瘤OS-732细胞系诱导血管生成作用及相关因子表达的研究

应用鸡胚绒毛尿囊膜模型（chick embryo chorioallantoic membrane,CAM),观察人骨肉瘤OS-732细胞系诱导血管生成过程及血管生长相关因子的表达。结果显示,本细胞系具有较强的促血管生成能力并表达血管内皮生长因子（vacular endothelial growth factor,VEGF),碱性成纤维细胞生长因子（basic fibroblast growth factor,bFGF0,鸡胚绒毛尿囊膜OS-732细胞系接种瘤细胞中血管内皮生长因子（VEGF）,转化生长因子β1(Transforming growth factor,TGF-β1）均呈阳性表达,而且VEGF呈持续高表达,结果表明VEGF,bFGF、TGF-β1可能共同参与骨肉瘤OS-732细胞系诱导的血管生成,而VEGF可能起着主要作用,提示阻断VEGF的作用可能影响骨肉瘤OS-732细胞系诱导的血管生成,此研究为以VEGF为靶点进行抗血管生成实验提供了依据。     

Moderate alcohol consumption induces sustained cardiac protection by activating PKC-epsilon and Akt

C57BL/6 mice were fed 18% ethanol (vol/vol) in drinking water for 12 wk. Isovolumic hearts were subjected to 20 min of ischemia and 30 min of reperfusion on a Langendorff apparatus. There were no differences in baseline hemodynamic function between hearts from ethanol (EtOH)-fed mice and controls. However, prior alcohol consumption doubled recovery of left ventricular developed pressure (68 +/- 8 vs. 33 +/- 8 mmHg for controls; n = 10, P < 0.05) and reduced creatine kinase release by half (0.26 +/- 0.04 vs. 0.51 +/- 0.08 U x min(-1) x g wet wt(-1) for controls; n = 10, P < 0.05). EtOH feeding doubled expression of activated protein kinase C epsilon (PKC)epsilon (n = 6, P < 0.05); whereas PKC inhibition blocked protection during ischemia-reperfusion. EtOH feeding also increased expression of Akt three- to fivefold (n = 6, P < 0.05), whereas PKC inhibition prevented increases in Akt kinase activity. We conclude that signaling pathways involving PKC-epsilon are critical for sustained EtOH-mediated cardioprotection and that Akt may be a downstream effector of resistance to myocardial reperfusion injury.     

Regulation of Vascular Endothelial Growth Factor Expression by cAMP in Rat Aortic Smooth Muscle Cells

Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen which stimulates angiogenesis. VEGF is regulated by multiple factors such as hypoxia, phorbol esters, and growth factors. However, data concerning the expression of VEGF in the different vascular cell types and its regulation by cAMP are not available. In the present study, we have investigated the effect of adenylate cyclase activation on VEGF mRNA expression in rat vascular cells in primary culture. Basal VEGF expression is greater in smooth muscle cells than in endothelial cells and fibroblasts. A 4-h treatment with forskolin (10⁻⁵M) induced a 2-fold stimulation of VEGF mRNA expression in smooth muscle cells and fibroblasts, but, in contrast, did not affect VEGF expression in endothelial cells. In smooth muscle cells, a pharmacologically induced increase in intracellular cAMP levels using iloprost or isoprenaline led to a rise in VEGF mRNA expression comparable to that induced by forskolin. Adenosine, which increases cAMP levels in smooth muscle cells, also increases VEGF expression. Moreover, the 2.2-fold stimulation of VEGF expression by adenosine was enhanced following a cotreatment with cobalt chloride (a hypoxia miming agent). The observed additive effect (4.3-fold increase) suggests that these two factors, hypoxia and adenosine, regulate VEGF mRNA expression in smooth muscle cells by independent mechanisms.     

Activation of the metabolic sensor-AMP activated protein kinase reverses impairment of angiogenesis in aging myocardial microvascular endothelial cells. Implications for the aging heart

Impairment of angiogenesis - new capillary blood vessel formation from pre-existing vessels, is frequent in aging tissues and cells. Reduced angiogenesis in aging individuals is associated with increased incidence of myocardial infarctions and other cardiovascular diseases. Therefore there is a need to develop novel strategies to enhance angiogenesis in aging individuals. Our previous study demonstrated aging-related impairment of angiogenesis in aging (vs. young) rat myocardial microvascular endothelial cells (MMEC), and identified reduced activation of the vascular endothelial growth factor (VEGF, the most potent stimulator of angiogenesis) gene as the main underlying mechanism. In the present study we examined the possibility of increasing angiogenesis and activating VEGF gene expression in aging MMECs using a chemical activator of the metabolic sensor - AMP activated protein kinase (AMPK). We hypothesized that activation of VEGF gene in aging MMECs by AMPK would stimulate angiogenesis and reverse the impairment in angiogenesis seen in these cells. We used MMECs isolated from aging (24 months old) Fisher F-344 rats and treated them with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), a specific pharmacological stimulator of AMPK. We examined: 1) in vitro angiogenesis; and 2) the expression of phosphorylated AMPK, VEGF, and P-MAPK/Erk1/2. Treatment of aging MMECs with AICAR increased in vitro angiogenesis and VEGF mRNA expression by 2.1-fold and 3.7-fold, respectively. Furthermore, AICAR treatment resulted in phosphorylation of MAPK/Erk1/2. This study demonstrated the successful use AICAR to reverse aging-related impairment of angiogenesis in aging MMECs by enhancing VEGF gene expression and also identified phosphorylation of MAPK/Erk1/2 as a likely mechanism of these changes.     

Exercise adaptation attenuates VEGF gene expression in human skeletal muscle

Angiogenesis is a component of the multifactoral adaptation to exercise training, and vascular endothelial growth factor (VEGF) is involved in extracellular matrix changes and endothelial cell proliferation. However, there is limited evidence supporting the role of VEGF in the exercise training response. Thus we studied mRNA levels of VEGF, using quantitative Northern analysis, in untrained and trained human skeletal muscle at rest and after a single bout of exercise. Single leg knee-extension provided the acute exercise stimulus and the training modality. Four biopsies were collected from the vastus lateralis muscle at rest in the untrained and trained conditions before and after exercise. Training resulted in a 35% increase in muscle oxygen consumption and an 18% increase in number of capillaries per muscle fiber. At rest, VEGF/18S mRNA levels were similar before (0.38 +/- 0.04) and after (1.2 +/- 0.4) training. When muscle was untrained, acute exercise greatly elevated VEGF/18S mRNA levels (16.9 +/- 6.7). The VEGF/18S mRNA response to acute exercise in the trained state was markedly attenuated (5.4 +/- 1.3). These data support the concept that VEGF is involved in exercise-induced skeletal muscle angiogenesis and appears to be subject to a negative feedback mechanism as exercise adaptations occur.     

Decreased expression of pigment epithelium derived factor (PEDF), an inhibitor of angiogenesis, in placentas of unexplained stillbirths

Normal placental vascular development depends upon the complex interactions between angiogenic inducers and inhibitors within the placenta. Alterations within the placental microenvironment can promote an imbalance in angiogenic mediators which may be associated with adverse perinatal outcomes. The purpose of this study was to investigate the placentas of infants with unexplained stillbirth as compared to live-born infants and to determine whether alterations in angiogenic inducer vascular endothelial growth factor (VEGF) or inhibitor pigment epithelium-derived factor (PEDF) are associated with altered angiogenesis, vascular remodeling and stillbirth. Placentas of 22 unexplained stillbirths and 44 age-matched live-born controls were scored for microvascular density (MVD), vasculopathy and microvascular permeability. A subset was scored for expression of angiogenic inducer VEGF and inhibitor pigment epithelium-derived factor. Stillborn placentas demonstrated higher MVD than controls (mean+SD: 116.6+/-46.3 v. 60.8+/-13.5, respectively, p<0.001). Vasculopathy was present in 10/22 (45%) stillbirths compared to 0/44 (0%) controls (p<0.001); increased vascular permeability was present in 15/22 (68%) cases and 5/44 (11%) controls (p<0.001). PEDF expression was significantly lower in stillborn placentas (1.7+/-0.3) than live-born controls (3.6+/-0.8, p<0.01) while VEGF expression was similar (3.3+/-0.7 v. 3.7+/-0.4, respectively, p>0.05). In conclusion, we found that unexplained stillbirth is associated with loss of angiogenic inhibitor PEDF, vasculopathy and heightened angiogenesis in the placenta.