Human Ang-2 Research Articles

Angiopoietin-2-related modulation of retinal angiogenesis – A new model for diabetic retinopathy

Diabetic retinopathy is the most prevalent cause of blindness among adults of working age. The earlies discernible lesion in the diabetic retina is pericyte loss. The Angiopoietin-Tie system plays a central role in this process. We examined the impact of retinal Angiopoietin 2 (Ang-2) overexpression on capillary cell cross-talk. We established and characterized a transgenic mouse which expresses human Ang-2 under control of a murin opsin promoter. Physiological sprouting of postnatal retinal vessels was examined in lectin-stained retinal whole mount preparations. Quantitative morphometry of retinal digest preparations was used to assess the endothelial/pericyte ratio as a measure of changes in the cellular composition of capillaries. The effect of retinal Ang-2 overexpression on pathological neovascularization was studied in the mouse model of hypoxia-induced proliferative retinopathy. Physiological sprouting was accelerated by Ang-2 overexpression, in association with a 17% deficit of pericytes in the deep capillary layers. MOpsinHAng-2 mice had significantly more retinal neovascularizations, both within and outside the retina, compared with controls. Taken together, our data suggest that Ang-2 is involved in the interaction between pericytes and endothelial cells, and may play an important role in the response-to-injury of the diabetic vessel system.

Protective Role of Angiopoietin-1 in Endotoxic Shock

Angiopoietin-1 (Ang1) plays an essential role in embryonic vasculature development, protects the adult peripheral vasculature from leakage, and has antiinflammatory properties. Because endotoxin-induced shock is a condition with microvascular leakage resulting from inflammation, we examined the potential therapeutic benefit of Ang1 in a murine model of lipopolysaccharide (LPS)-induced endotoxic shock. To induce endotoxic shock, LPS was injected intraperitoneally into C57BL/6 mice. Half of the mice received an intravenous application of 1.0x10(9) plaque-forming units of an adenoviral construct expressing human Ang1 (AdhAng1); in the other half an identical vector expressing green fluorescent protein (AdGFP) was injected as a control. In the AdhAng1-treated mice, hepatic transfection and high expression of circulating Ang1 protein were observed. Whereas in LPS-treated control mice, hemodynamic function was severely depressed 12 hours after LPS injection (decrease of blood pressure from 91+/-3 to 49+/-7 mm Hg, dP/dt(max) from 7284+/-550 to 2699+/-233 mm Hg/s, cardiac output from 11.3+/-1.2 to 2.8+/-0.8 mL/min; P<0.0005), in LPS-treated AdhAng1 mice blood pressure fell only to 76+/-3 mm Hg, dP/dt(max) to 5091+/-489 mm Hg/s, and cardiac output to 6.7+/-1.4 mL/min (P<0.05). This resistance to LPS-induced hemodynamic changes was reflected by an improved Kaplan-Meier survival rate of the AdhAng1 mice. Histological analysis revealed that lung injury after LPS injection was markedly attenuated in AdhAng1 mice. In addition, LPS-induced increase in lung water content and pulmonary myeloperoxidase activity was significantly reduced. Furthermore, LPS-induced increases in the expression level of vascular cell adhesion molecule-1, intracellular adhesion molecule-1, and E-selectin protein in the lungs were markedly lower in AdhAng1 mice than in control mice. Finally, in the mice overexpressing Ang1, pulmonary endothelial NO synthase (eNOS) expression and activity remained preserved after LPS challenge, providing evidence that the beneficial effect of Ang1 in endotoxic shock is mediated by eNOS-derived NO. Our study demonstrates an improved mortality rate in mice with endotoxic shock pretreated with an adenoviral construct encoding Ang1. The enhanced survival rate induced by Ang1 was accompanied by an improvement in hemodynamic function, reduced lung injury, a lower expression of inflammatory adhesion molecules, and preserved eNOS activity in the lung tissue. Ang1 may therefore have utility as an adjunctive agent for the treatment of septic shock condition.

Expression and purification of recombinant human angiopoietin-2 produced in Chinese hamster ovary cells

Angiopoietin-2 (Ang2) is a complex regulator of vascular remodeling that plays a role in both blood vessel sprouting and blood vessel regression through its receptor Tie2. Recombinant Chinese hamster ovary (rCHO) cell lines expressing a high level (20 μg/mL) of recombinant human Ang2 protein (rhAng2) with an amino-terminal FLAG-tag was constructed by transfecting the expression vectors into dihydrofolate reductase (dhfr)-deficient CHO cells and the subsequent gene amplification in medium containing stepwise increments in methotrexate level such as 0.02, 0.08, and 0.32 μM. The rhAng2 secreted from rCHO cells was purified at a purification yield of 53.6% from the cultured medium using an anti-FLAG M2 agarose affinity gel. SDS–PAGE and Western blot analyses showed that rCHO cells secret rhAng2 as a homodimeric glycoprotein form. Furthermore, rhAng2 binds to the Tie2 receptor and phosphorylates Tie2 in a concentration-dependent manner. Therefore, our rhAng2 could be useful for clarifying biological effect of exogenous Ang2 in the future.

Expression of angiopoietin-2 in endothelial cells is controlled by positive and negative regulatory promoter elements.

Angiopoietin-2 (Ang-2) is a non-signal transducing ligand of the endothelial receptor tyrosine kinase Tie-2. Ang-2 is produced by endothelial cells and acts as an autocrine regulator mediating vascular destabilization by inhibiting Angiopoietin-1-mediated Tie-2 activation. To examine the transcriptional regulation of Ang-2, we studied the Ang-2 promoter in endothelial cells and nonendothelial cells. The human Ang-2 promoter contains a 585-bp region around the transcriptional start site (-109 to +476) that is sufficient to control endothelial cell-specific and cytokine-dependent Ang-2 expression. Strong repressor elements of Ang-2-promoter activity are located in the 5'-region of the promoter and in the first intron. The Ets family transcription factors Ets-1 and Elf-1 act as strong enhancers of endothelial cell Ang-2-promoter activity. Ets-binding sites -4 and -7 act as positive regulators, whereas Ets-binding site -3 acts as negative regulator. Demethylation experiments revealed that the Ang-2 gene (in contrast to the Tie-2 gene) is not controlled by imprinting. The data determine unique positive and negative regulatory mechanisms of endothelial cell Ang-2 expression and provide further evidence for the critical role of Ang-2 as a key autocrine regulator of vascular stability and responsiveness.

189. Sendai Virus Mediated Angiopoietin-1 Gene Therapy for Cardiovascular Diseases

Background: Sendai virus (SeV) is negative-stranded RNA virus, which is classified as a type I parainfluenza virus belonging to the family of Paramyxoviridae. SeV has been known as rodent respiratory virus, however it is considered to be non-pathogen for humans. Recently, efficient gene transduction into various types of cells and tissue has been reported. Angiopoietin-1 (Ang1) is a critical angiogenic factor for vascular maturation and enhances vascular endothelial growth factor (VEGF)-induced angiogenesis in a complementary manner. Previously we reported Ang1 gene therapy clearly promoted myocardial angiogenesis and prevented remodeling in a rat AMI model. In the present study, we created recombinant Sendai virus vector (SeVV) encoding human Ang1 and evaluated therapeutic effect in the cardiovascular diseases. We also evaluated the angiogenic effect of mesenchymal stem cells (MSCs) transplantation with SeVV-mediated Ang1 gene modification.

Transcriptional regulation of human angiopoietin-2 by transcription factor Ets-1

Angiopoietin-2 (Ang-2) plays an important role in destabilizing vessels for angiogenesis, however its transcriptional regulation has not been determined. Here we isolated about 3.2 kb of the 5 ′ upstream of the human Ang-2 gene and further characterized the transcriptional regulation of Ang-2. We found 10 Ets binding sites (EBSs) in this region. Ets-1 and Ets-2 but not Erg-1 augmented its promoter activity. Sequential deletion of EBSs and mutation analysis revealed that EBS8 located at −416/−406 from the translation initiation site was critical for Ets-1- or Ets-2-stimulated promoter activity. VEGF induced the expression of Ets-1 and Ang-2 in human umbilical vein endothelial cells (HUVECs). Electrophoretic mobility shift and chromatin immunoprecipitation assays showed that Ets-1 bound to EBS8 in HUVECs. Moreover, synthetic double-strand oligonucleotides containing corresponding EBS8 abrogated the induction of Ang-2 by VEGF in HUVECs. These results indicate that Ets-1 and the corresponding EBS are critical for the induction of Ang-2.

Single chain Fv antibody against angiopoietin-2 inhibits VEGF-induced endothelial cell proliferation and migration in vitro

Angiopoietin-2 (Ang2) promotes tumor growth and metastasis by specifically priming endothelial cells for angiogenesis. Multiple angiogenic factors up-regulate expression of Ang2, suggesting that Ang2 may be the common pathway in growth factor initiated-angiogenesis. Using phage display technology, we generated single chain Fv molecule against human Ang2 (scFv–Ang2) with high affinity ( K d =0.01 μ M) from a mouse phage antibody library. Compared with control scFv, the mouse scFv–Ang2 completely inhibited the proliferation of human umbilical vein endothelial cells (HUVECs) treated with vascular endothelial growth factor (VEGF, 10 ng/ml), but not that of the cells treated with either basic fibroblast growth factor, or angiotensin II, or Ang2. Chemotaxis assay showed that scFv–Ang2 could block completely Ang2-induced (100%) and partially VEGF-induced (49%) migration of HUVECs. The results indicate that Ang2 takes part in the VEGF-induced angiogenesis and scFv–Ang2 might be a promising compound in blocking both VEGF and Ang2 induced angiogenesis.

Angiopoietin-1 protects against the development of cardiac allograft arteriosclerosis.

Angiopoietin (Ang)-1 is an angiogenic growth factor that counteracts the permeability and proinflammatory effects of vascular endothelial growth factor and other proinflammatory cytokines. Recently, we demonstrated that vascular endothelial growth factor enhances cardiac allograft arteriosclerosis. Here, we studied the roles of Ang1, its natural antagonist Ang2, and their receptor Tie2 in rat cardiac allograft arteriosclerosis. Heterotopic cardiac allografts and syngrafts were transplanted from Dark Agouti (DA) to Wistar-Furth rats and from DA to DA rats, respectively. Immunohistochemistry disclosed that only a few mesenchymal cells expressed Ang1 in normal hearts and syngrafts, whereas no immunoreactivity was found in cardiac allografts undergoing chronic rejection. Ang2 and Tie2 immunoreactivity was induced mainly in capillaries and postcapillary venules in chronic allografts when compared with syngeneic controls, but no immunoreactivity was found in arterial endothelium. Intracoronary perfusion of cardiac allografts with a clinical-grade adenoviral vector encoding human Ang1 (Ad.Ang1) protected against the development of allograft arteriosclerosis. Ad.Ang1 perfusion reduced Ang2 expression in microcirculation, the numbers of graft-infiltrating leukocytes, and the level of immunoactivation and interstitial fibrosis, as well as both the incidence and intensity of intimal lesions. Ad.Ang1 perfusion also increased CD34+ stem cell counts in peripheral blood. Our findings suggest that the antiinflammatory properties of Ang1 may offer an entirely new therapeutic approach to prevent cardiac allograft arteriosclerosis.

Biological action of angiopoietin-2 in a fibrin matrix model of angiogenesis is associated with activation of Tie2.

The endothelial cell (EC) specific tyrosine kinase receptor, Tie2, interacts with at least two ligands, angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2). Ang1 stimulates Tie2 receptor autophosphorylation, while Ang2 has been reported to inhibit Ang1-induced Tie2 receptor autophosphorylation. We studied the effects of Ang1 and Ang2 in an in vitro model of angiogenesis. Human ECs (HUVEC), cultured on 3-D fibrin matrices, were treated with conditioned media (CM) from stably transfected cells expressing human Ang1 or Ang2, or with purified recombinant proteins. EC tube formation was measured as a differentiation index (DI), calculated as the ratio of total tube length over residual of EC monolayer. CM from Ang1 overexpressing A10 SMC or HEK293T cells induced profound HUVEC differentiation, resulting in the formation of extensive capillary-like tubes within 48 h (DI: 24.58+/-5.91 and 19.13+/-7.86, respectively) vs. control (DI: 2.73+/-1.68 and 2.15+/-1.45, respectively, both P<0.001). Interestingly, CM from two independent cell lines overexpressing Ang2 also produced a significant increase in EC differentiation (DI: 9.22+/-3.00 and 9.72+/-4.84, both P<0.005 vs. control) although the degree of angiogenesis was significantly less then that seen with Ang1. Addition of Ang1* (a genetically engineered variant of naturally occurring Ang1) or Ang2 also resulted in dose dependent increases in DI, which were blocked by an excess of soluble Tie2 receptor (20 microg/ml). Both Ang1* and Ang2 induced modest increases in [3H]thymidine incorporation into HUVECs (20 and 26%, respectively), which were inhibited by excess soluble Tie2. Although Ang2 was unable to induce significant Tie2 receptor phosphorylation during a 5-min exposure, a 24-h pretreatment with Ang2, followed by brief re-exposure, produced Tie2 phosphorylation in HUVEC comparable to that produced by Ang1*. These results demonstrate for the first time that Ang2 may have a direct role in stimulating Tie2 receptor signaling and inducing in vitro angiogenesis. Our findings suggest that the physiological role of Ang2 is more complex than previously recognized: acting alternately to promote or blunt Tie2 receptor signaling in endothelial cells, depending on local conditions.