A nuclease-resistant RNA aptamer specifically inhibits angiopoietin-1-mediated Tie2 activation and function

Tie2 is a receptor tyrosine kinase that is expressed predominantly in the endothelium and plays key roles in both physiological and pathological angiogenesis. The ligands for Tie2, the angiopoietins (Ang), perform opposing functions in vascular maintenance and angiogenesis; Ang1 regulates vascular quiescence, while Ang2 is thought to promote vascular destabilization and facilitate angiogenesis. However, the mechanisms responsible for these differences are not understood. To begin to elucidate the molecular differences between the angiopoietins, we previously developed a specific RNA aptamer inhibitor of Ang2. Here, we used the same iterative in vitro selection process, termed SELEX (Systematic Evolution of Ligands by EXponential enrichment), to screen a library of 2′-fluoro-modified ribonucleotides for Ang1-binding aptamers. After nine rounds of selection, we identified a single clone, ANG9-4, that bound with high affinity to human Ang1 (K _d 2.8 nM) but not Ang2 (K _d  > 1 μM), demonstrating specificity for Ang1. ANG9-4 blocked Ang1-mediated Tie2 phosphorylation and downstream Akt activation. Moreover, ANG9-4 inhibited Ang1-induced endothelial cell survival. Together, these findings demonstrate the feasibility of developing an Ang1-inhibitory aptamer. ANG9-4 and its derivatives may provide useful tools for elucidating the biology of Ang1 and for treating certain angiogenic diseases.     

The venous graft as an effector of early angiogenesis in a fibrin matrix

The arteriovenous loop (AV loop) model is gaining importance as a means of initiating and sustaining perfusion in tissue engineering constructs in vivo. This study represents an attempt to dissect the morphology of early arterialization and angiogenesis in the AV loop in a fibrin matrix with special focus on the interpositional venous graft (IVG) segment. An AV loop was constructed in 30 rats using the femoral vessels and an IVG. The AV loop was encased in an isolation chamber filled with a fibrin matrix. Evaluation methods included scanning electron microscopy (SEM) of corrosion casts, immune histology and micro magnetic resonance angiography (MRA). Direct luminal neovascular sprouting was evident between day 10 and day 14 from the vein and the IVG but not from the arterial segment. Arterialization of the IVG manifested itself on the corrosion casts as a gradual reduction in luminal caliber with onset after day 7. Microdissection of the microvascular replicas could demonstrate for the first time the presence of direct luminal sprouts from the IVG. MRA was used to display the shunt pattern of perfusion in the patent AV loop. From the three segments of the vascular axis in the AV loop the IVG is the most versatile for applications in the clinical as well as the experimental setting. Kinetics of angiogenesis warrant further investigation in the IVG.     

Neurohormonal activation is associated with increased levels of plasma matrix metalloproteinase-2 in human heart failure.

AIMS: Development of heart failure depends on systemic and molecular abnormalities among which are the activation of neurohormonal systems and the increase of matrix metalloproteinases (MMPs). This study assessed the relationship between catecholamines and active MMPs in vivo in patients with severe congestive heart failure (CHF) and in vitro in human cardiac fibroblasts. METHODS AND RESULTS: Forty patients with CHF due to dilated cardiomyopathy, either idiopathic (n=20) or secondary to ischaemic heart disease (n=20), were compared with 20 healthy subjects. Plasma MMP-2 and MMP-9 activity, but not TIMP-2, were significantly higher in patients than in controls (median MMP-2, 270 vs. 214 ng/mL, P=0.006; MMP-9 16.3 vs. 8.7 ng/mL, P<0.0001). Similarly, noradrenaline, but not adrenaline, was significantly higher in patients (noradrenaline 645 vs. 157 pg/mL, P<0.0001; adrenaline 86.0 vs. 72.6 pg/mL, P=0.68). No difference in any parameter was observed between patient groups. The intra-group correlation between MMP-2 and noradrenaline was significant (r=0.33, P=0.01); indeed, noradrenaline appear to be a predictor of MMP-2. Moreover, this catecholamine increased MMP-2 in human cardiac fibroblasts. CONCLUSIONS: The positive correlation between noradrenaline and MMP-2 in severe CHF patients, together with the in vitro induction of MMP-2 by this catecholamine, suggests a potential biochemical link between noradrenaline and MMP-2.     

血管生成素1基因上调Tie2受体表达促进大鼠急性心肌梗死血管生成的实验研究

目的　通过观察血管生成素 1 (angiopoietin 1 ,Ang1 )基因对急性心肌梗死大鼠心肌Tie2受体表达的影响 ,探讨Ang1基因治疗促进血管生成 ,减少梗死面积的可能机制。方法　结扎左前降支冠状动脉制备大鼠心肌梗死模型 ,心肌内分别直接注射空载质粒或phAng1质粒 ;分别于术后第 3,7,1 4 ,2 8天取材 ,利用RT PCR技术分析心肌组织内Tie2受体表达 ;免疫组化方法计算血管生成以及肌性小动脉生成数量 ,Masson染色法检测心肌梗死面积。结果　Ang1基因治疗组Tie2受体mRNA表达明显高于对照组 ,于术后 7天达到高峰 ,2 8天降至正常水平。术后第 7,1 4和 2 8天 ,Ang1基因治疗组的血管生成及肌性小动脉生成较同期对照组均明显增加 ,梗死面积减小。结论　Ang1基因心肌内注射促进急性心肌梗死血管生成及肌性小动脉生成 ,减小梗死面积 ,其机制可能与促进Tie2受体上调有关。     

FGF-2 binding to fibrin(ogen) is required for augmented angiogenesis

We have shown previously that fibrin(ogen) binds fibroblast growth factor 2 (FGF-2) and potentiates stimulation of endothelial-cell (EC) proliferation. We have now used 2 FGF-2 mutants differing only in the 5 residues constituting the binding site to characterize the importance of this interaction in angiogenesis. The nonbinding (2212) and binding (221*2) mutants stimulated EC proliferation by 2.2 +/- 0.4-fold and 2.9 +/- 0.3-fold over control, respectively, and both were similar to wild-type (wt) FGF-2 (2.5 +/- 0.3-fold). Proliferation was augmented by fibrinogen to 5.3 +/- 1.2-fold and 4.8 +/- 0.8-fold with wtFGF-2 and 221*2, whereas no augmentation occurred with 2212 and fibrinogen. Using a placental explant model in a fibrin matrix, wtFGF-2 resulted in 2.6 +/- 0.9-fold more growth over control, and 221*2 increased growth 3.3 plus or minus 0.9-fold. Vessel outgrowth with 2212 was minimal and comparable to control. Similarly, fibrinogen potentiated wtFGF-2 or 221*2-mediated angiogenesis in the chicken chorioallantoic membrane model. In a mouse Matrigel implant model, fibrinogen significantly increased angiogenesis with either wtFGF-2 or 221*2, whereas there was no augmentation with 2212. These results demonstrate that binding of FGF-2 to fibrin(ogen) mediated by the 5-residue FGF-2-fibrin(ogen) interactive site is required for augmented angiogenesis.     

Inhibition of rat corneal angiogenesis by a nuclease-resistant RNA aptamer specific for angiopoietin-2

Angiopoietin-2 (Ang2) appears to be a naturally occurring antagonist of the endothelial receptor tyrosine kinase Tie2, an important regulator of vascular stability. Destabilization of the endothelium by Ang2 is believed to potentiate the actions of proangiogenic growth factors. To investigate the specific role of Ang2 in the adult vasculature, we generated a nuclease-resistant RNA aptamer that binds and inhibits Ang2 but not the related Tie2 agonist, angiopoietin-1. Local delivery of this aptamer but not a partially scrambled mutant aptamer inhibited basic fibroblast growth factor-mediated neovascularization in the rat corneal micropocket angiogenesis assay. These in vivo data directly demonstrate that a specific inhibitor of Ang2 can act as an antiangiogenic agent.     

Reactivation of proliferin gene expression is associated with increased angiogenesis in a cell culture model of fibrosarcoma tumor progression

Proliferin (PLF) is an angiogenic placental hormone. We now report that PLF gene expression can also occur in a progressive fibrosarcoma mouse tumor cell model. PLF mRNA and protein are detectable at very low levels in cell lines derived from the mild noninvasive stage of tumor development. Expression is greatly augmented in cell lines from the aggressively invasive stage of development, a stage at which the tumor becomes highly angiogenic, and PLF expression remains high in cell lines from the end stage of fibrosarcoma. Activator protein 1 factors present at high levels in the more invasive stages of the tumor may in part allow for increased PLF expression, as cells from the mild stage in which c-jun and junB are stably expressed secrete levels of PLF comparable to that of the advanced stages. Secreted PLF protein is functionally important in tumor cell angiogenic activity, as demonstrated by the reduction of angiogenic activity in fibrosarcoma cell culture medium by immunodepletion of PLF. These results suggest that an extraembryonic genetic program, which has evolved to support fetal growth, may be reactivated in certain tumors and contribute to tumor growth.     

Roles of the receptor tyrosine kinases Tie1 and Tie2 in mediating the effects of angiopoietin-1 on endothelial permeability and apoptosis

Angiopoietin-1 (Ang1) has key roles in development and maintenance of the vascular system. The ligand is a potent inhibitor of vascular leakage and suppresses endothelial apoptosis and vessel regression. Ang1 was originally identified as a ligand for the receptor tyrosine kinase Tie2. Recently however Ang1 has also been found to activate the related tyrosine kinase Tie1. The contribution of Tie1 to mediating the effects of Ang1 on endothelial function is not known. In this study we used an siRNA approach to investigate the relative importance of Tie1 and Tie2 in transducing the effects of Ang1 on monolayer permeability and induction of apoptosis in human endothelial cells. siRNA directed against either Tie1 or Tie2 suppressed expression of each respective receptor by more than 90%. Ang1 inhibited endothelial monolayer permeability and this effect was prevented by suppression of Tie2 expression. In contrast, Ang1 inhibition of permeability was not affected by suppression of Tie1 expression. The ability of Ang1 to inhibit induction of apoptosis in response to serum deprivation was completely blocked by suppression of Tie2 expression, but not diminished by suppression of Tie1 expression. Taken together these data demonstrate that Tie2 mediates the inhibitory effects of Ang1 on endothelial permeability and apoptosis. The data also demonstrates that Tie1 does not transduce anti-apoptotic or anti-permeability effects of Ang1 in endothelial cells.     

Membrane-type matrix metalloproteinase-mediated angiogenesis in a fibrin-collagen matrix

Adult angiogenesis, associated with pathologic conditions, is often accompanied by the formation of a fibrinous exudate. This temporary matrix consists mainly of fibrin but is intermingled with plasma proteins and collagen fibers. The formation of capillary structures in a fibrinous matrix in vivo was mimicked by an in vitro model, in which human microvascular endothelial cells (hMVECs) seeded on top of a fibrin-10% collagen matrix form capillarylike tubular structures after stimulation with basic fibroblast growth factor/tumor necrosis factor alpha (bFGF/TNF-alpha) or vascular endothelial growth factor (VEGF)/TNF-alpha. In the fibrin-collagen matrix the metalloproteinase inhibitor BB94 inhibited tubule formation by 70% to 80%. Simultaneous inhibition of plasmin and metalloproteinases by aprotinin and BB94 caused a nearly complete inhibition of tubule formation. Adenoviral transduction of tissue inhibitor of metalloproteinases 1 (TIMP-1) and TIMP-3 into endothelial cells revealed that TIMP-3 markedly inhibited angiogenesis, whereas TIMP-1 had only a minor effect. Immunohistochemical analysis showed the presence of matrix metalloproteinase 1 (MMP-1), MMP-2, and membrane-type 1 (MT1)-MMP, whereas MMP-9 was absent. The endothelial production of these MMPs was confirmed by antigen assays and real-time polymerase chain reaction (PCR). MT1-MMP mRNA was markedly increased in endothelial cells under conditions that induced tubular structures. The presence of MMP-1, MMP-2, and MT1-MMP was also demonstrated in vivo in the newly formed vessels of a recanalized arterial mural thrombus. These data suggest that MMPs, in particular MT-MMPs, play a pivotal role in the formation of capillarylike tubular structures in a collagen-containing fibrin matrix in vitro and may be involved in angiogenesis in a fibrinous exudate in vivo.     

Periodontitis is associated with platelet activation

There is an epidemiological association between periodontitis and cardiovascular disease (CVD). In periodontitis, low grade systemic inflammation and bacteremia occur regularly. Such events may contribute to platelet activation and subsequent pro-coagulant state. This study aimed to investigate platelet activation in periodontitis patients. The study is composed of two parts. In the first part, plasma levels of soluble(s) P-selectin and sCD40 ligand were measured as general markers of platelet activation in periodontitis patients (n=85) and in healthy controls (n=35). In the second part, surface-exposed P-selectin and the ligand-binding conformation of the glycoprotein IIb-IIIa complex (binding of PAC-1 antibody) were determined on individual platelets in whole blood of periodontitis patients (n=18) and controls (n=16). Patients had significantly elevated plasma levels of sP-selectin (P<0.001) and increased binding of PAC-1 on isolated platelets (P=0.033). Platelet activation was more pronounced in the patients with more severe periodontal disease, showing a severity-dependence. The levels of sCD40 ligand and of platelet-bound P-selectin were not increased. Periodontitis is associated with increased platelet activation. Since platelet activation contributes to a pro-coagulant state and constitutes a risk for atherothrombosis, platelet activation in periodontitis may partly explain the epidemiological association between periodontitis and CVD.     

Plasma ratio of angiopoietin-2 to angiopoietin-1 is a biomarker of vascular impairment in chronic obstructive pulmonary disease patients

Chronic obstructive pulmonary disease (COPD) patients have an increased risk of cardiovascular disease. Muscle biopsies have revealed that the muscle vasculature in COPD patients was characterized by a capillary rarefaction with reduced pericyte coverage. Thus, an imbalance of the plasma Angiopoietin-1 / Angiopoietin-2 (Ang2/Ang1) ratio could constitute a non-invasive marker of the muscle vascular impairment. In 14 COPD patients (65.5±5.1-year-old) and 7 HC (63.3±5.8-year-old), plasma samples were obtained at 3 time-points: before, after 5 weeks (W5), and after 10 weeks (W10) of exercise training. COPD patients showed a muscle capillary rarefaction at baseline with a reduced capillary coverage at W5 and W10. The plasma Ang2/Ang1 ratio was significantly higher in COPD patients vs. HC during the training (Group: p=0.01). The plasma Ang2/Ang1 ratio was inversely correlated with the pericyte coverage index regardless of the time period W0 (r=?0.51; p=0.02), W5 (r=?0.48; p=0.04), and W10 (r=?0.61; p<0.01). Last, in ECFC/MSC co-cultures exposed to the W10 serum from COPD patients and HC, the plasma Ang2/Ang1 at W10 were inversely correlated with calponin staining (r=?0.64. p=0.01 and r= 0.71. p<0.01, Fig. 1B), in line with a role of this plasma Ang2/Ang1 in the MSC differentiation into pericytes. Altogether, plasma Ang2/Ang1 ratio could constitute a potential marker of the vascular impairment in COPD patients.

     

Embryonic stem cell tumor model reveals role of vascular endothelial receptor tyrosine phosphatase in regulating Tie2 pathway in tumor angiogenesis

Inhibiting angiogenesis has become an effective approach for treating cancer and other diseases. However, our understanding of signaling pathways in tumor angiogenesis has been limited by the embryonic lethality of many gene knockouts. To overcome this limitation, we used the plasticity of embryonic stem (ES) cells to develop a unique approach to study tumor angiogenesis. Murine ES cells can be readily manipulated genetically; in addition, ES cells implanted subcutaneously in mice develop into tumors that contain a variety of cell types (teratomas). We show that ES cells differentiate into bona fide endothelial cells within the teratoma, and that these ES-derived endothelial cells form part of the functional tumor vasculature. Using this powerful and flexible system, the Angiopoietin/Tie2 system is shown to have a key role in the regulation of tumor vessel size. Endothelial differentiation in the ES teratoma model allows gene-targeting methods to be used in the study of tumor angiogenesis.