Angiopoietin-1 reduces iopromide-induced endothelial cell apoptosis through activation of phosphatidylinositol 3'-kinase/p70 S6 kinase.

Radiocontrast media can induce vascular endothelial cell apoptosis. Apoptotic damage to the vascular endothelium is an important mechanism in vascular disease. Several growth factors with anti-apoptotic effects may help protect the vascular endothelium from apoptosis. The present study evaluated whether the radiocontrast agent iopromide induces apoptosis in human umbilical vein endothelial cells and also whether angiopoietin-1 (Ang1) protects against iopromide-induced apoptosis through the p70 S6 kinase-dependent signaling pathway. Iopromide induced apoptosis in vascular endothelial cells in a dose-dependent manner. Ang1 reduced iopromide-induced apoptosis in a dose-dependent manner. Wortmannin and LY294002, phosphatidylinositol 3'-kinase inhibitors, decreased the Ang1-induced anti-apoptotic effect. Ang1 mediates the activation of mTOR/ribosomal protein p70 S6 kinase through phosphatidylinositol-3' kinase. Wortmannin and rapamycin, an inhibitor of mTOR, suppressed Ang1-induced p70 S6 kinase phosphorylation and partially inhibited the Ang1-induced anti-apoptotic effect. These results suggest that Ang1 may protect vascular endothelial cells from iopromide-induced apoptosis through phosphatidylinositol 3'-kinase and mTOR/S6 kinase. Pretreatment with Ang1 could help maintain normal vascular endothelial cell integrity before and during systemic radiocontrast administration.

Angiopoietin-1 reduces VEGF-stimulated leukocyte adhesion to endothelial cells by reducing ICAM-1, VCAM-1, and E-selectin expression.

Vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang1) are potent vasculogenic and angiogenic factors that hold promise as a means to produce therapeutic vascularization and angiogenesis. However, VEGF also acts as a proinflammatory cytokine by inducing adhesion molecules that bind leukocytes to endothelial cells, an initial and essential step toward inflammation. In the present study, we used human umbilical vascular endothelial cells (HUVECs) to examine the effect of Ang1 on VEGF-induced expression of three adhesion molecules: intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin. Interestingly, Ang1 suppressed VEGF-induced expression of these adhesion molecules. Furthermore, Ang1 reduced VEGF-induced leukocyte adhesion to HUVECs. These results demonstrate that Ang1 counteracts VEGF-induced inflammation by reducing VEGF-induced endothelial adhesiveness.

The angiopoietin-tie2 system in coronary artery endothelium prevents oxidized low-density lipoprotein-induced apoptosis.

OBJECTIVES: A healthy, intact coronary artery endothelium is important because most common coronary artery diseases result from loss of endothelial integrity. In this study, we explored the biological significance of the angiopoietin-Tie2 system in porcine coronary artery. METHODS: Cultured porcine coronary artery endothelial cells and explanted coronary arteries were used. RESULTS: Immunohistochemical analyses indicated that Ang1 is selectively expressed in vascular muscular cells, whereas angiopoietin-2 (Ang2) and Tie2 are selectively expressed in endothelial cells. Accordingly, Ang1 mRNA is mainly expressed in cultured porcine coronary artery vascular smooth muscle cells, whereas Ang2 and Tie2 mRNAs are mainly expressed in cultured porcine coronary artery endothelial cells (PCAECs). Ang1 (200 ng/ml) induced Tie2 phosphorylation, while Ang2 (200 ng/ml) did not produce Tie2 phosphorylation. Ang1 increased the survival of cultured PCAECs during apoptosis induced by oxidized low-density lipoprotein (OxLDL). This survival effect was does-dependent and PI. Furthermore, Ang1 also protected endothelial cells of explanted coronary artery against OxLDL-induced apoptosis artery. CONCLUSION: These results suggest that adult coronary artery contains Ang1-Tie2 components that enhance endothelial cell survival to help maintain the normal integrity of the coronary artery endothelium.

Vascular endothelial growth factor expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin through nuclear factor-kappa B activation in endothelial cells.

Vascular endothelial growth factor (VEGF) induces adhesion molecules on endothelial cells during inflammation. Here we examined the mechanisms underlying VEGF-stimulated expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin in human umbilical vein endothelial cells. VEGF (20 ng/ml) increased expression of ICAM-1, VCAM-1, and E-selectin mRNAs in a time-dependent manner. These effects were significantly suppressed by Flk-1/kinase-insert domain containing receptor (KDR) antagonist and by inhibitors of phospholipase C, nuclear factor (NF)-kappaB, sphingosine kinase, and protein kinase C, but they were not affected by inhibitors of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) 1/2 or nitric-oxide synthase. Unexpectedly, the phosphatidylinositol (PI) 3'-kinase inhibitor wortmannin enhanced both basal and VEGF-stimulated adhesion molecule expression, whereas insulin, a PI 3'-kinase activator, suppressed both basal and VEGF-stimulated expression. Gel shift analysis revealed that VEGF stimulated NF-kappaB activity. This effect was inhibited by phospholipase C, NF-kappaB, or protein kinase C inhibitor. VEGF increased VCAM-1 and ICAM-1 protein levels and increased leukocyte adhesiveness in a NF-kappaB-dependent manner. These results suggest that VEGF-stimulated expression of ICAM-1, VCAM-1, and E-selectin mRNAs was mainly through NF-kappaB activation with PI 3'-kinase-mediated suppression, but was independent of nitric oxide and MEK. Thus, VEGF simultaneously activates two signal transduction pathways that have opposite functions in the induction of adhesion molecule expression. The existence of parallel inverse signaling implies that the induction of adhesion molecule expression by VEGF is very finely regulated.

Angiopoietin-2 at high concentration can enhance endothelial cell survival through the phosphatidylinositol 3'-kinase/Akt signal transduction pathway.

The angiopoietin-Tie2 system in endothelial cells is an important regulator of vasculogenesis and vascular integrity. High levels of angiopoietin-2 (Ang2) mRNA are observed in vascular activation during tumorigenesis. Although Ang2 is known to be a naturally occurring antagonist of angiopoietin-1 (Ang1) in vivo, the exact function of Ang2 itself is not known. Here, we found that a high concentration of Ang2 (800 ng/ml) acts as an apoptosis survival factor for endothelial cells during serum deprivation apoptosis. The survival effect of high concentration Ang2 was blocked by pre-treatment with soluble Tie2 receptor and the PI 3'-kinase-specific inhibitors, wortmannin and LY294002. Accordingly, 800 ng/ml of Ang2 induced phosphorylation of Tie2, the p85 subunit of phosphatidylinositol 3'-kinase (PI 3'-kinase), and serine-threonine kinase Akt at Ser473 in the human umbilical vein endothelial cells; lower concentrations of Ang2 (50 - 400 ng/ml) did not produce notable effects. These findings indicate that at high concentrations, Ang2, like Ang1, can be an apoptosis survival factor for endothelial cells through the activation of the Tie2 receptor, PI 3'-kinase and Akt, and thus may be a positive regulator of tumor angiogenesis. Oncogene (2000) 19, 4549 - 4552.

Angiopoietin-1 induces endothelial cell sprouting through the activation of focal adhesion kinase and plasmin secretion.

Angiopoietin-1 (Ang1) is a strong inducer of endothelial cell sprouting, which is a first step in both angiogenesis and neovascularization. We examined the mechanisms underlying Ang1-induced cell sprouting using porcine pulmonary artery endothelial cells. Ang1 induced the nondirectional and directional migration of endothelial cells mediated through the Tie2 but not the Tie1 receptor. Ang1 induced tyrosine phosphorylation of p125(FAK), and this phosphorylation was dependent on phosphatidylinositol (PI) 3'-kinase activity. Ang1 induced the secretion of plasmin and matrix metalloproteinase-2 (MMP-2), which is inhibited by PI 3'-kinase inhibitors. Ang1 also induced the secretion of small amounts of proMMP-3 and proMMP-9 but not proMMP-1. Ang1 suppressed the secretion of tissue inhibitor of metalloproteinase-2 (TIMP-2), but not of TIMP-1. Addition of alpha(2)-antiplasmin, a combination of TIMP-1 and TIMP-2, or PI 3'-kinase inhibitors inhibited Ang1-induced sprouting activity. Therefore, Ang1-induced sprouting activity in endothelial cells may be accomplished by cytoskeletal changes and secretion of proteinases and may be largely mediated through intracellular PI 3'-kinase activation.

Molecular cloning, expression, and characterization of angiopoietin-related protein. angiopoietin-related protein induces endothelial cell sprouting.

Using degenerate polymerase chain reaction, we isolated a cDNA encoding a novel 493-amino acid protein from human and mouse adult heart cDNAs and have designated it angiopoietin-related protein-2 (ARP2). The NH(2)-terminal and COOH-terminal portions of ARP2 contain the characteristic coiled-coil domain and fibrinogen-like domain that are conserved in angiopoietins. ARP2 has two consensus glycosylation sites and a highly hydrophobic region at the NH(2) terminus that is typical of a secretory signal sequence. Recombinant ARP2 expressed in COS cells is secreted and glycosylated. In human adult tissues, ARP2 mRNA is most abundant in heart, small intestine, spleen, and stomach. In rat embryos, ARP2 mRNA is most abundant in the blood vessels and skeletal muscles. Endothelial and vascular smooth muscle cells also contain ARP2 mRNA. Recombinant ARP2 protein induces sprouting in vascular endothelial cells but does not bind to the Tie1 or Tie2 receptor. These results suggest that ARP2 may exert a function on endothelial cells through autocrine or paracrine action.

Changes in the expression of c-myc, RB and tyrosine-phosphorylated proteins during proliferation of NIH 3T3 cells induced by hyaluronic acid.

We have shown that hyaluronic acid stimulates the proliferation of quiescent NIH 3T3 cells. We have shown that treatment of 1 mg/ml hyaluronic acid results in increase of tyrosine phosphorylation of two proteins, MW 124 kDa and 60 kDa as detected by anti-tyrosine antibodies by Western blot analysis. Maximum phosphorylation occurred within 2 h after addition of 1 mg/ml hyaluronic acid. Stimulation of proliferation was also accompanied by increase in c-Myc protein, which was inhibited by amlloride, an inhibitor of Na+/H+ antiporter and EGTA and increase in the steady state level of pRb, the RB gene product. These results suggest that the intracellular signal transduction pathways that mediate the stimulatory effects of hyaluronic acid on cellular proliferation are similar to those of growth factors.

Phorbol esters potentiate tyrosine phosphorylation of epidermal growth factor receptors in A431 membranes by a calcium-independent mechanism.

Incubation of membranes prepared from A431 cells with either epidermal growth factor (EGF) or phorbol 12-myristate 13-acetate (PMA) stimulates the transfer of 32phosphate from [gamma-32P]ATP into 8-10 membrane proteins. The major phosphorylated protein migrates on NaDodSO4/polyacrylamide gels with an apparent Mr of 180,000, corresponding to the previously identified EGF receptor. Stimulation of EGF receptor phosphorylation by PMA does not require Ca2+, suggesting that prior activation of protein kinase C is not a prerequisite for phosphate transfer. PMA-enhanced phosphorylation proceeds at 4 degrees C and requires Mn2+, both properties of tyrosine-specific protein kinases. Phospho amino acid analysis of the Mr 180,000 receptor band shows that only tyrosine residues are phosphorylated when A431 membranes are treated with either EGF or PMA. Moreover, proteolysis reveals that these residues are located in the same peptides of the receptor. These results demonstrate that a potent tumor-promoting phorbol ester can mimic a critical early response usually elicited by EGF.