2-Methoxyestradiol (2-ME) is an endogenous metabolite of estradiol-17beta and the oral contraceptive agent 17-ethylestradiol. 2-ME was recently reported to inhibit endothelial cell proliferation. The current study was undertaken to explore the mechanism of 2-ME effects on endothelial cells, especially whether 2-ME induces apoptosis, a prime mechanism in tissue remodeling and angiogenesis. Cultured bovine pulmonary artery endothelial cells (BPAEC) exposed to 2-ME showed morphological (including ultrastructural) features characteristic of apoptosis: cell shrinkage, cytoplasmic and nuclear condensation, and cell blebbing. 2-ME-induced apoptosis in BPAEC was a time- and concentration-dependent process (EC50 = 0.45 +/- 0.09 microM, n = 8). Nucleosomal DNA fragmentation in BPAEC treated with 2-ME was identified by agarose gel electrophoresis (DNA ladder) as well as in situ nick end labeling. Under the same experimental conditions, estradiol-17beta and two of its other metabolites, estriol and 2-methoxyestriol (< or =10 microM), did not have an apoptotic effect on BPAEC. 2-ME activated stress-activated protein kinase (SAPK)/c-Jun amino-terminal protein kinase in BPAEC in a concentration-dependent manner. The activity of SAPK was increased by 170 +/- 27% and 314 +/- 22% over the basal level in the presence of 0.4 and 2 microM 2-ME (n = 3-6), respectively. The activation of SAPK was detected at 10 min, peaked at 20 min, and returned to basal levels at 60 min after exposure to 2-ME. Inhibition of SAPK/c-Jun amino-terminal protein kinase activation by basic fibroblast growth factor, insulin-like growth factor, or forskolin reduced 2-ME-induced apoptosis. Immunohistochemical analysis of BPAEC indicated that 2-ME up-regulated expression of both Fas and Bcl-2. In addition, 2-ME inhibited BPAEC migration (IC50 = 0.71 +/- 0.11 microM, n = 4) and basic fibroblast growth factor-induced angiogenesis in the chick chorioallantoic membrane model. Taken together, these results suggest that promotion of endothelial cell apoptosis, thereby inhibiting endothelial cell proliferation and migration, may be a major mechanism by which 2-ME inhibits angiogenesis.

Chemokines play an important role in the regulation of endothelial cell (EC) function, including proliferation, migration and differentiation during angiogenesis, and re-endothelialization after injury. In this study, reverse transcriptase-polymerase chain reaction was used to reveal expression of various CXC and CC chemokine receptors in human umbilical vein EC. Northern analysis showed that CXCR4 was selectively expressed in vascular EC, but not in smooth muscle cells. Compared with other chemokines, stromal cell-derived factor-1␣ (SDF-1␣), the known CXCR4 ligand, was an efficacious chemoattractant for EC, causing the migration of ϳ40% input cells with an EC 50 The vascular endothelium is strategically located to play a prominent sensory and effector cell role in the maintenance of hemostasis, and during the vascular response to inflammation, infection, and injury (1, 2). The endothelium is also integrally associated with angiogenesis (3) and cardiovascular disorders such as atherosclerosis and restenosis (4). Endothelial cells (EC) 1 interact with various inflammatory cells, as well as platelets and smooth muscle cells via a variety of chemotactic factors such as chemokines and their receptors (5, 6). Chemokines are classified into at least two groups, which differ with respect to the organization of the dicysteine motif present at the NH 2 terminus. The ␣-chemokines, characterized by the CXC motif include PF-4, IL-8, ␥IP-10 and SDF-1. The ␤-chemokines, characterized by the CC motif include MCP-1, MIP-1␣ and 1␤, and RANTES (5, 7, 8). Chemokines mediate their specific effect on target cells through two related subfamilies of G-protein coupled receptors. To date, several CXC and CC functional human chemokine receptors have been discovered (9 -16). In line with their well defined role as mediators of diapedesis, the chemokine receptors have been primarily localized on neutrophils, monocytes, lymphocytes, and eosinophils (5). However, little is known about other distinct functions of these cytokines and their interaction with non-hematopoietic cells.Three lines of evidence indicate that human EC also express the genes for chemokine receptors and thus play an active and important role as target cells for chemokine function. First, the proliferation, migration, and differentiation of vascular EC, during angiogenesis, is modulated by chemokines, apparently via specific receptors. Thus, IL-8 is an inducer of angiogenesis (17), whereas PF-4 (18 -20), Gro-␤ (21), and ␥IP-10 (22) are inhibitors of EC proliferation and angiogenesis. Second, it has been suggested that leukocyte adhesion to the endothelium and transmigration require that chemotactic factors be immobilized on the EC surface (23,24). This idea is necessitated due to the obvious conceptual difficulty in generating a chemotactic gradient of soluble chemokines under conditions of blood flow. Although chemokines can bind cell surface proteoglycans (24,25), vascular endothelium may still require expression of receptors that are capable of immobilizing chemokin...

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Abstract. Modulation of endothelial cell proliferation and cell cycle progression by the "chemokine" platelet factor-4 (PF-4) was investigated. PF-4 inhibited DNA synthesis, as well as proliferation of endothelial cells derived from large and small blood vessels. Inhibition by PF-4 was independent of the type and the concentration of stimuli used for the induction of endothelial cell proliferation. Inhibition of cell growth by PF-4 was reversible. The effects of PF-4 were antagonized by heparin. Cell cycle analysis using [3H]thymidine pulse labeling during traverse of synchronous cells from G0/G1 to S phase revealed that addition of PF-4 during G1 phase completely abolished the entry of cells into S phase. In addition, PF-4 also inhibited DNA synthesis in cells that were already in S phase. In exponentially growing cells, addition of PF-4 resulted in an accumulation of >70 % of the cells in early S phase, as determined by FACS ® (BectonDickinson Immunocytometry Systems, Mountain View, CA). In cells synchronized in S phase by hydroxyurea and then released, addition of PF-4 promptly blocked further progression of DNA synthesis. These results demonstrate that in GO/Gl-arrested cells, PF-4 inhibited entry of endothelial cells into S phase. More strikingly, our studies have revealed a unique mode of endothelial cell growth inhibition whereby PF-4 effectively blocked cell cycle progression during S phase.

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