Mayumi Abe

Ets‐1 regulates angiogenesis by inducing the expression of urokinase‐type plasminogen activator and matrix metalloproteinase‐1 and the migration of vascular endothelial cells

The coordinate induction of protease activities and cell migration is a principal feature of endothelial cells (ECs) invading the interstitial space in the initial step of angiogenesis. However, the molecular mechanisms of these events are not fully characterized. Ets‐1 is a member of the ets gene family of transcription factors, which binds to the Ets binding motif in the cis‐acting elements and regulates the expression of certain genes. Four typical angiogenic growth factors, aFGF, bFGF, VEGF, and EGF, induced the expression of ets‐1 mRNA in either human umbilical vein endothelial cells (HUVECs), ECV‐304 cells (immortalized HUVECs), or human omental microvascular endothelial cells (HOMECs). The expression of ets‐1 reached its maximum at 2 hr after factor addition and then decreased to the basal level by 12 hr. For characterization of the role of Ets‐1 in angiogenesis, ets‐1 antisense and sense oligodeoxynucleotides (ODNs) were constructed. The ets‐1 antisense ODN but not sense ODN efficiently blocked the synthesis of Ets‐1 protein by human ECs in response to angiogenic growth factors. Moreover, the ets‐1 antisense ODN but not sense ODN almost completely abolished the binding of endothelial cell extract to DNA containing the Ets binding motif. The expression of urokinase‐type plasminogen activator and matrix metalloproteinase‐1 and the migration of ECs in response to growth factors were significantly inhibited by ets‐1 antisense ODN but not by sense ODN. Tube formation by HOMECs in type 1 collagen gel stimulated with EGF was abrogated by ets‐1 antisense ODN. Finally, the expression of Ets‐1 protein in ECs during angiogenesis in vivo was confirmed by an immunohistochemical analysis using a murine angiogenesis model. These results indicate that the induction of ets‐1 mRNA is a mutual phenomenon in ECs stimulated with angiogenic growth factors. Ets‐1 appears to play an important role in angiogenesis, regulating the expression of proteases and the migration of ECs.

Roles of two VEGF receptors, Flt-1 and KDR, in the signal transduction of VEGF effects in human vascular endothelial cells.

Vascular endothelial growth factor (VEGF) is a principal regulator of vasculogenesis and angiogenesis. VEGF expresses its effects by binding to two VEGF receptors, Flt-1 and KDR. However, properties of Flt-1 and KDR in the signal transduction of VEGF-mediated effects in endothelial cells (ECs) were not entirely clarified. We investigated this issue by using two newly developed blocking monoclonal antibodies (mAbs) against Flt-1 and KDR. VEGF elicits DNA synthesis and cell migration of human umbilical vein endothelial cells (HUVECs). The pattern of inhibition of these effects by two mAbs indicates that DNA synthesis is preferentially mediated by KDR. In contrast, the regulation of cell migration by VEGF appears to be more complicated. Flt-1 regulates cell migration through modulating actin reorganization, which is essential for cell motility. A distinct signal is generated by KDR, which influences cell migration by regulating cell adhesion via the assembly of vinculin in focal adhesion plaque and tyrosine-phosphorylation of focal adhesion kinase (FAK) and paxillin.

ETS-1 converts endothelial cells to the angiogenic phenotype by inducing the expression of matrix metalloproteinases and integrin β3

The transcription factor ETS‐1 is induced in endothelial cells (ECs) by angiogenic growth factors and the specific elimination of ETS‐1 synthesis by antisense oligodeoxynucleotide inhibited angiogenesis in vitro (Iwasaka et al., 1996, J Cell Physiol 169:522–531). To understand the precise role of ETS‐1 in angiogenesis, we established both high and low ETS‐1 expression EC lines and compared angiogenic properties of these cell lines with those of the parental murine EC line, MSS‐31. Although growth rate was almost identical for each cell line, the invasiveness was markedly enhanced in high ETS‐1 expression cells and reduced in low ETS‐1 expression cells compared with that of parental cells. The gene expressions of matrix metalloproteinases (MMP‐1, MMP‐3, and MMP‐9) and gelatinolytic activity of MMP‐9 were significantly increased in high ETS‐1 expression cells. Low ETS‐1 expression cells could not spread on a vitronectin substratum, and the phosphorylation of focal adhesion kinase was markedly impaired because of the reduced expression of integrin β3. These results indicate that ETS‐1 is a principal regulator that converts ECs to the angiogenic phenotype. J Cell Physiol 178:121–132, 1999.

Establishment of a Simple and Practical Procedure Applicable to Therapeutic Angiogenesis

BACKGROUND Therapeutic angiogenesis is thought to be beneficial for serious ischemic diseases. This investigation was designed to establish a simple and practical procedure applicable to therapeutic angiogenesis. METHODS AND RESULTS When cultured skeletal muscle cells were electrically stimulated at a voltage that did not cause their contraction, vascular endothelial growth factor (VEGF) mRNA was augmented at an optimal-frequency stimulation. This increase of VEGF mRNA was derived primarily from transcriptional activation. Electrical stimulation increased the secretion of VEGF protein into the medium. This conditioned medium then augmented the growth of endothelial cells. The effect of electrical stimulation was further confirmed in a rat model of hindlimb ischemia. The tibialis anterior muscle in the ischemic limb was electrically stimulated. The frequency of stimulation was 50 Hz and strength was 0.1 V, which was far below the threshold for muscle contraction. After a 5-day stimulation, there was a significant increase in blood flow within the muscle. Immunohistochemical analysis revealed that VEGF protein was synthesized and capillary density was significantly increased in the stimulated muscle. Rats tolerated this procedure very well, and there was no muscle contraction, muscle injury, or restriction in movement. CONCLUSIONS We propose this procedure as a simple and practical method of therapeutic angiogenesis.

Roles of Extracellular Signal-regulated Kinase 1/2 and p38 Mitogen-activated Protein Kinase in the Signal Transduction of Basic Fibroblast Growth Factor in Endothelial Cells during Angiogenesis

We examined the role of mitogen‐activated protein (MAP) kinases in the signal transduction of basic fibroblast growth factor (bFGF)‐mediated effects in endothelial cells (ECs). When MSS31 murine endothelial cells were stimulated with bFGF, three MAP kinase homologs, extracellular signal‐regulated kinase (ERK) 1/2, c‐Jun N‐terminal kinase (JNK) 1, and p38 MAP kinase were activated. The inhibition of the ERK1/2 pathway with PD98059, a specific inhibitor of MEK1, or of the p38 MAP kinase pathway with SB203580, a specific inhibitor of p38 MAP kinase, abrogated bFGF‐mediated tube formation by MSS31 cells in type I collagen gel. Tube formation in type I collagen gel requires proliferation and migration of these cells, and degradation of the extracellular matrix by these cells. Both PD98059 and SB203580 inhibited bFGF‐stimulated DNA synthesis as well as migration of MSS31 cells. Cell migration requires cytoskeleton reorganization and cell adhesion. bFGF induced actin reorganization and vinculin assembly in the focal adhesion plaque, both of which were inhibited by SB203580 but not by PD98059. bFGF induced the expression of the transcription factor ETS‐1 in MSS31 cells. ETS‐1 is responsible for the expression of proteases as well as integrin β3 subunit in ECs, and converts ECs to invasive phenotype. PD98059 inhibited this induction of ETS‐1, whereas SB203580 did not. These results indicate that ERK1/2 and p38 MAP kinase are requisite for the signal transduction of bFGF in ECs. The roles of these two MAP kinase homologs are not identical, but these kinases work in a coordinated fashion.

cDNA microarray analysis of the gene expression profile of VEGF-activated human umbilical vein endothelial cells.

Vascular endothelial growth factor (VEGF) is one of the most important factors that stimulate angiogenesis and vascular permeability. To clarify the role of VEGF, we analysed a human cDNA chip containing 7267 human genes to identify genes induced by VEGF in human umbilical vein endothelial cells (HUVECs). One hundred thirty-nine cDNAs, including ninety-nine previously known and forty poorly characterized or novel sequences, were increased more than two-fold by VEGF within twenty-four hours of stimulation. Among them, only five are known to regulate angiogenesis: cyclooxygenase 2 (COX2), heparin-binding epidermal growth factor-like growth factor, early growth response 1 (EGR 1), CYR61, and angiopoietin 2. Fifty-three genes induced within the first two hours were thought to be directly induced by VEGF. Of these, Down syndrome candidate region 1 (maximum induction = 6.1-fold) was the most profoundly induced, followed by Mif1 (KIAA0025; 5.5-fold), COX2 (4.7-fold), EGR 3 (3.7-fold), EGR 2 (3.2-fold), bactericidal/permeability-increasing protein (3.1-fold), and CD1B antigen, b polypeptide (3.1-fold). In addition to the genes mentioned above, there were many poorly characterized or novel genes induced by VEGF. Further analysis of these genes may aid in the elucidation of the molecular mechanisms of angiogenesis or vascular permeability stimulated by VEGF.

Activation of Latent Transforming Growth Factor ß

Publisher Summary This chapter discusses the activation of latent TGF-β1 receptors for members of the TGF-β family appear to have a ubiquitous distribution and these growth factors can affect the physiology of many cell types. The fact that TGF-β is secreted as a latent molecule is well established. It is also evident that a critical step in the regulation of TGF-β activity is the release of active growth factor from the latent precursor. The conditions under which this activation occurs and its mechanism remain largely undefined. Insight into the extracellular regulation of TGF-β activity may be important for several reasons: (1) Investigators have far been unable to apply a comprehensive functional definition to TGF-β. (2) The prudent clinical use of TGF-β demands an understanding of the extracellular regulation of latent TGF- β. Such understanding may allow minimizing effects on nontarget organs. (3) Understanding the regulation of TGF activity may allow to effectively interfere with inappropriate TGF-β activity, such as that implicated in hepatic and pulmonary fibrosis and glomerulonephritis. To date, it is unclear whether there is a single or several mechanisms for latent TGF- β activation. Further investigation is needed to determine the relative significance of the various activation mechanisms that have been demonstrated in vivo and to dissect the complex interactions that appear to be involved in the activation of latent TGF- β.

Properties of Two VEGF Receptors, Flt-1 and KDR, in Signal Transductiona

Abstract: The properties of two VEGF receptors, Flt‐1 and KDR, in the signal transduction of VEGF in human umbilical vein endothelial cells (HUVECs) were investigated by using two newly developed blocking monoclonal antibodies (mAbs) against Flt‐1 and KDR. VEGF stimulated the expression of transcription factor Ets‐1 as well as matrix metalloproteinase‐1 (MMP‐1) and Flt‐1 in HUVECs. The KDR/Flt‐1 heterodimer and the KDR homodimer mediate the expression of Ets‐1, MMP‐1, and Flt‐1. VEGF also stimulated DNA synthesis and migration of HUVECs. DNA synthesis is mediated by the same signaling system as the expression of Ets‐1. In contrast, cell migration is regulated by two distinct signaling systems. The Flt‐1 homodimer is required for actin reorganization. The KDR/Flt‐1 heterodimer and the KDR homodimer are required for the assembly of vinculin in focal adhesion plaque by regulating the phosphorylation of focal adhesion kinase (FAK) and paxillin.

INDUCTION OF ETS-1 IN ENDOTHELIAL CELLS DURING REENDOTHELIALIZATION AFTER DENUDING INJURY

Ets‐1, a transcription factor, is induced in endothelial cells (ECs) during angiogenesis. Here, we investigated the expression of Ets‐1 during reendothelialization. When a confluent monolayer of human umbilical vein endothelial cell line, ECV304, was denuded, ECV304 at the wound edge expressed Ets‐1. An immunohistochemical analysis revealed that Ets‐1 accumulated in migrating cells at the wound edge and returned to basal level when reendothelialization was accomplished. This induction of Ets‐1 could be reproduced in in vivo denudation of rat aortic endothelium by a balloon catheter. The induction of Ets‐1 in ECs after denudation was regulated transcriptionally, and humeral factors released from injured ECs might not be responsible. Mitogen‐activated protein kinase (MAPK) activities were investigated to explore the mechanism of this induction. Although extracellular signal‐regulated protein kinase 1/2 (ERK1/2), c‐Jun N‐terminal kinase 1 (JNK1), and p38 were activated after denudation, the activation of ERK1 and p38 was more rapid and prominent. PD98059, a specific MAPK/ERK kinase (MEK) 1 inhibitor, did not affect the induction of ets‐1 mRNA, whereas SB203580, a specific p38 inhibitor, almost completely abrogated its induction. These results indicate that Ets‐1 is induced in ECs after denudation through activation of p38. This induction of Ets‐1 may be relevant for reendothelialization by regulating the expression of certain genes. J. Cell. Physiol. 176:235–244, 1998.

Platelet factor 4 blocks the binding of basic fibroblast growth factor to the receptor and inhibits the spontaneous migration of vascular endothelial cells

Platelet factor 4 (PF-4) blocked the binding of basic fibroblast growth factor (bFGF) to the plasma membrane receptor. Five micrograms/ml of PF-4 completely blocked the specific binding of bFGF to the receptor of NIH 3T3 cells. Endogenously produced bFGF regulates the spontaneous migration of bovine aortic endothelial (BAE) cells as an autocrine factor (Sato and Rifkin, 1988). PF-4 inhibited the spontaneous migration of BAE cells in a reversible and dose dependent manner. The inhibition reached maximum at 5 micrograms/ml of PF-4, where the binding of bFGF to the receptor was completely blocked.