Both Ets-1 and GATA-1 are essential for positive regulation of platelet factor 4 gene expression

In the rat platelet factor 4 (PF4) promoter, Ets motifs and GATA motifs are located at positions -880, -75 and -135, -30, respectively, and their motifs are found in the promoter region of most megakaryocyte protein genes. In order to investigate how the Ets and GATA motifs affect PF4 promoter activity, we constructed Ets and/or GATA motif mutant genes. A single disruption of either -75Ets, -135GATA, or -30GATA significantly reduced PF4 promoter activity, and double disruptions involving these motifs completely abolished it. Furthermore, gel-retardation assays revealed that Ets-1 and GATA-1 proteins from HEL and MEG-01 cells bound to the Ets motifs and GATA motifs, respectively. Co-transfection experiments showed that the overexpression of Ets-1 and/or GATA-1 enhanced the expression of the PF4 promoter reporter gene. These effects of Ets-1 and GATA-1 on PF4 promoter activity are additive. When HEL cells were treated with dimethylsulfoxide in order to induce differentiation into megakaryocytes, the mRNA level of ets-1 increased 10-fold, which might be directly correlated with the significant increase in PF4 mRNA level induced by dimethylsulfoxide. All these results strongly suggest that both Ets-1 and GATA-1 play key roles in the positive regulation of PF4 gene expression.     

Inhibition of vascular endothelial growth factor-induced endothelial cell migration by ETS1 antisense oligonucleotides

Vascular endothelial growth factor (VEGF) increased the level of ETS1 mRNA in human umbilical vein endothelial cells (HUVEC) and human lung microvascular endothelial cells (HMVEC-L) over 5-fold. Protein levels were shown to increase concordantly. VEGF was also found to stimulate the invasiveness of endothelial cells as measured by migration through Matrigel- or gelatin-coated membranes. The VEGF-induced invasiveness was inhibited by ETS1 antisense oligonucleotides but not by a sense control. In addition, the ETS1 antisense oligonucleotides reduced the levels of ETS1 and urokinase-type plasminogen activator mRNAs. The antisense oligonucleotides directed against the ETS1 gene thus altered a cellular property of endothelial cells that is correlated with the ability of the cells to migrate through basement membranes. Together, these observations demonstrate a direct role for the ETS1 gene in angiogenesis.     

Proliferative hemangiomas: analysis of cytokine gene expression and angiogenesis

Hemangiomas are benign vascular tumors of childhood that can lead to disfigurement and/or life-threatening consequences. The pathogenesis of hemangioma formation is likely to involve increased angiogenesis. Basic fibroblast growth factor and vascular endothelial growth factor are cytokines that stimulate angiogenesis in multiple in vivo and in vitro models. Proliferative hemangiomas have been found to have elevated levels of basic fibroblast growth factor and vascular endothelial growth factor protein, but the gene expression of these cytokines in human specimens has not been previously studied. We examined the gene expression and spatial distribution of basic fibroblast growth factor and vascular endothelial growth factor messenger RNA in proliferative versus involuted human hemangioma specimens using nonisotopic in situ hybridization techniques. Thirteen hemangioma specimens were harvested during initial surgical excision. In situ hybridization was performed on frozen sections of both proliferative and involuted hemangioma specimens using genetically engineered antisense probes specific for basic fibroblast growth factor and vascular endothelial growth factor messenger RNA. Controls were an interleukin-6 sense sequence and a transforming growth factor-beta 1 antisense sequence. A large number of cells within the specimens of proliferative hemangiomas revealed localized gene expression of basic fibroblast growth factor and vascular endothelial growth factor messenger RNA (626 +/- 129 and 1660 +/- 371 cells/mm2, respectively). The majority of the cells were endothelial in origin. In contrast, involuted hemangioma specimens revealed significantly lower numbers of cells staining positive for basic fibroblast growth factor and vascular endothelial growth factor messenger RNA (44 +/- 11 and 431 +/- 76 cells/mm2, respectively; p < 0.05). Transforming growth factor-beta 1 messenger RNA was slightly more expressed by involuted hemangiomas (117 +/- 30 cells/mm2). There were very low levels of transforming growth factor-beta 1 gene expression from proliferative hemangiomas (37 +/- 24 cells/mm2; p < 0.02). These data demonstrate that (1) in situ hybridization allows identification and relative quantitation of cells expressing messenger RNA for specific growth factors in human hemangioma specimens; (2) basic fibroblast growth factor and vascular endothelial growth factor messenger RNA are up-regulated in proliferative hemangiomas; and (3) transforming growth factor-beta 1 messenger RNA remains low in both proliferative and involuted hemangiomas. Because basic fibroblast growth factor and vascular endothelial growth factor messenger RNA have been implicated in the pathobiology of human hemangioma formation, biochemical modulation of these angiogenic cytokines may eventually help inhibit proliferation and promote regression of hemangiomas.     

Angiogenic activity of osteopontin-derived peptide SVVYGLR

Angiogenesis plays an important role in various pathological conditions as well as some physiological processes. Although a number of soluble angiogenic factors have been reported, extracellular matrix also has crucial effect on angiogenesis through interaction with endothelial cells. Since recent reports showed osteopontin had some angiogenic activity, the effect of the SVVYGLR peptide, novel binding motif in osteopontin molecule, on angiogenesis was examined in this study. Synthetic peptide SVVYGLR did not have proliferative effect on endothelial cells but adhesion and migration activity to endothelial cells. Furthermore, SVVYGLR had as potent activity for tube formation in three-dimensional collagen gel as vascular endothelial growth factor which is known to be the strongest angiogenic factor. Electron microscopical analysis showed a number of microvilli on the endothelial luminar surface and tight junction formation in the luminar intercellular border between endothelial cells, indicating SVVYGLR induced cell porarity and differentiation of endothelial cells. This small peptide might be expected to stimulate angiogenesis to improve some ischemic conditions in the future because of some advantages due to smaller molecular weight.     

Angiogenesis: new aspects relating to its initiation and control

Angiogenesis, the formation of new microvessels from parent microvessels, involves remodeling the basement membrane and interstitial extracellular matrix (ECM) using degrading proteases produced by the endothelial cells (ECs) and other adjacent cells, and the synthesis of ECM molecules by these cells. Degraded ECM releases previously bound heparin-binding cytokines (and growth factors) which are able to act as ligands to high-affinity receptors on various target cells, including ECs. The EC carries receptors for a number of cytokines which are produced by neighboring cells or released from the ECM and which can either induce or suppress the angiogenic phenotype of the EC. ECs are able to synthesize and secrete cytokines with auto- and paracrine effects. Angiogenesis, which virtually never occurs physiologically in adult tissues (except in the ovary, the endometrium and the placenta), is essential in wound healing and inflammation. Angiogenesis is, in fact, strictly controlled by a redundancy of pro- and anti-angiogenic paracrine peptide molecules, some of which have recently been described. The expression and synthesis of two distinct anti-angiogenic factors is, for example, controlled by the p53 tumor suppressor gene. In certain hypoxic conditions, chronic inflammatory diseases and syndromes, angiogenesis is of pathogenic and prognostic significance. Angiogenesis is, moreover, essential for the growth and metastatic spread of solid tumors. This indicates the potential for developing new therapeutic strategies not only for tumors but also in diseases such as rheumatoid arthritis, psoriasis, liver cirrhosis and diabetic retinopathy. Moreover, the therapeutic induction of angiogenesis in ischemic tissues using recombinant cytokines is also promising for clinical application. In fact, the first successful human gene therapy for stimulating angiogenesis has recently been reported.     

Human fibroblasts downregulate plasminogen activator inhibitor type-1 in cultured human macrovascular and microvascular endothelial cells

We have previously reported that plasminogen activator inhibitor type-1 (PAI-1) expression in endothelial cells (ECs) can be modulated differently by smooth muscle cells depending on their origin. Human pulmonary artery smooth muscle cells (HPASMCs) strongly downregulated PAI-1 expression in ECs. Fibroblasts (FBs) are another cell type that could come in close contact with ECs. Therefore, it was the aim of this study to investigate whether FBs could also influence the fibrinolytic potential of ECs. As in the case of HPASMCs, PAI-1 antigen produced by human umbilical vein ECs (HUVECs) cocultured with human skin FBs (HSFBs) was significantly lower as compared with the sum of PAI-1 secreted by the respective cell types cultured separately. Not only HUVECs but also human skin microvascular ECs (HSMECs) responded in a dose-dependent way to serum-free conditioned media (CM) from HSFBs from one individual donor. Similar results were obtained when CM from HSFBs from four other individual donors were used. PAI-1 mRNA decreased in HUVECs incubated for 6 hours with HSFB-CM to 24% to 55% of control, depending on the preparation of HSFBs used. A significant PAI-1 downregulatory effect was only observed when CM from low-passage HSFBs (up to passage no. 5) was used, whereas no reduction in EC PAI-1 production was observed with CM obtained from HSFBs in passage no. 8. This PAI-1 downregulatory activity present in HSFB-CM was heat-labile and had a molecular mass of approximately 5 kD. When CM from HPASMCs was analyzed in the same way, an almost identical elution profile was found. In conclusion, our data showed that FBs can decrease the expression of PAI-1 in ECs. Such an effect could be operative during wound-healing and at other capillary sites where FBs could render ECs profibrinolytic, thereby facilitating processes requiring an increase in proteolytic activity such as EC migration and proliferation.     

Effects of NMDA-R1 antisense oligodeoxynucleotide administration: behavioral and radioligand binding studies

The effects of an antisense phosphodiester oligodeoxynucleotide (ODN) directed to the NR1 subunit of the NMDA receptor mRNA and of its corresponding sense ODN were investigated in mice. Treatment with the antisense ODN significantly increased the time mice spent in the open arms of an elevated maze while the total number of arm entries was unaltered. Furthermore, seizure latencies after the administration of an ED100 dose of NMDA (150 mg/kg) were significantly higher in antisense treated animals compared to vehicle controls. At the same time, treatment with NR1 antisense ODN significantly reduced the Bmax of [3H]CGS-19755 binding (2101 fmol/mg protein) compared to both vehicle (2787 fmol/mg protein) and sense (2832 +/- 39 fmol/mg protein) controls without any significant change in KD (33 nM). A corresponding reduction of [3H]CGP-39653 binding was also observed after treatment with NR1 antisense compared to both sense and vehicle controls. In contrast, neither antisense nor sense ODNs altered the proportion of high affinity glycine sites or the potency of glycine at either high or low affinity glycine binding sites to inhibit [3H]CGP-39653 binding. These results show that in vivo treatment with NR1 antisense ODNs to the NMDA receptor complex reduces antagonist binding at NMDA receptors and has pharmacological effects similar to those observed with some NMDA receptor antagonists. These results also suggest that treatment with antisense ODNs may provide another means to investigate allosteric modulation of receptor subtypes in vivo.     

Significance of vessel count and vascular endothelial growth factor and its receptor (KDR) in intestinal-type gastric cancer

Angiogenesis is essential for tumor growth and metastasis and depends on the production of angiogenic factors by host and/or tumor cells. The role of angiogenesis and angiogenic factor expression in intestinal- and diffuse-type gastric cancer are undefined. Archival specimens of 51 intestinal-type and 38 diffuse-type human gastric carcinomas were examined for tumor vessel counts, angiogenic factor expression, and the presence or absence of angiogenic factor receptors on tumor endothelium using antibodies against vascular endothelial growth factor (VEGF) and its receptors (KDR and flt-1), basic fibroblast growth factor (bFGF) and its receptors (bek and flg), and factor VIII (endothelial cells). Vessel count and VEGF and bFGF expression were higher in intestinal-type than in diffuse-type gastric cancers (P = 0.01, P < 0.001, and P < 0.001, respectively). Similarly, vessel count and VEGF expression were higher in patients with liver metastasis than in patients with peritoneal dissemination (P = 0.003 and P = 0.01, respectively). Vessel count correlated with VEGF expression and the presence of endothelial KDR in intestinal-type gastric cancer (P = 0.003 and P = 0.02, respectively) but not diffuse-type gastric cancer. Vessel count, VEGF expression, and presence of endothelial KDR increased with increasing stage of disease in intestinal-type gastric cancer but not diffuse-type gastric cancer. The expression of bFGF and its receptors did not correlate with vessel count in either cancer type. These findings suggest that the pattern of metastasis in intestinal-type gastric cancer is angiogenesis dependent. The correlation of VEGF expression and its endothelial receptor with vessel count and stage of disease suggests that VEGF is at least one of the factors responsible for the induction of angiogenesis in intestinal-type gastric cancer.