Endothelial progenitor cells as putative targets for angiostatin.

Angiostatin, a product of the proteolytic cleavage of plasminogen, possesses potent antitumor and antiangiogenic properties in vivo. Studies with cultured endothelial cells suggest that under certain conditions, angiostatin inhibits the migration and proliferation of these cells or, alternatively, increases their rate of apoptosis. In general, the effects of angiostatin have been considerably less potent in vitro than in vivo. One potential explanation for this disparity is that the in vivo target of angiostatin is not the mature endothelial cell. Recently, evidence has accumulated to show that circulating endothelial progenitor cells (EPCs) contribute to neovascularization. In this study, we have isolated EPCs from human subjects and demonstrated that, in contrast to that of mature endothelial cells, the growth of EPCs is exquisitely sensitive to angiostatin. These results suggest that angiostatin and related compounds may exert their biological effects by inhibiting the contribution of EPCs to angiogenesis and not by altering the growth of mature endothelial cells.

A role for reactive oxygen species in endothelial cell anoikis.

When adherent cells, such as epithelial or endothelial cells, are detached and continuously maintained in suspension, they undergo a form of programmed cell death termed anoikis. We demonstrate that coincident with endothelial cell detachment, there is a dramatic rise in the intracellular level of reactive oxygen species (ROS). Reattachment to a solid surface rapidly attenuates the level of ROS. The mitochondria appear to be the major source of the detachment-induced rise in ROS. The change in the intracellular redox state appears to contribute to endothelial anoikis, because treatment with either the cell-permeant antioxidant N-acetylcysteine or the flavin protein inhibitor diphenylene iodonium is demonstrated to reduce oxidant levels and protect against subsequent cell death. Similarly, the endogenous intracellular level of ROS is shown to correlate with the extent of cell death. Finally, we demonstrate that the activities of both caspases and of the c-Jun N-terminal kinases are modulated by the rise in intracellular ROS levels. These results suggest that oxidants serve as signaling molecules and regulators of anoikis.

Regulation of endothelial cell adherens junctions by a Ras-dependent signal transduction pathway.

Adherens junctions, consisting of transmembrane cadherin molecules and their associated cytoplasmic alpha-, beta-, and gamma-catenin proteins, are thought to be critical for the development of stable cell adhesion and subsequent 3-dimensional tissue organization. In human endothelial cells there is a marked induction of gamma-catenin levels when cells reach confluence. We demonstrate that expression of a dominant negative ras gene product (N17ras) via adenoviral mediated gene transfer inhibits the confluent-dependent rise in gamma-catenin mRNA and protein levels. Consistent with its effects on overall gamma-catenin levels, expression of N17ras also reduces the amount of gamma-catenin associated with the adherens junction. Finally, although expression of N17ras under normal culture conditions produces no clear morphological phenotype, endothelial cells expressing a dominant negative ras gene product fail to form 3-dimensional, vascular-like structures when plated on reconstituted extracellular matrix.

A requirement for the rac1 GTPase in the signal transduction pathway leading to cardiac myocyte hypertrophy.

We have used adenoviral-mediated gene transfer of a constitutively active (V12rac1) and dominant negative (N17rac1) isoform of rac1 to assess the role of this small GTPase in cardiac myocyte hypertrophy. Expression of V12rac1 in neonatal cardiac myocytes results in sarcomeric reorganization and an increase in cell size that is indistinguishable from ligand-stimulated hypertrophy. In addition, V12rac1 expression leads to an increase in atrial natriuretic peptide secretion. In contrast, expression of N17rac1, but not a truncated form of Raf-1, attenuated the morphological hypertrophy associated with phenylephrine stimulation. Consistent with the observed effects on morphology, expression of V12rac1 resulted in an increase in new protein synthesis, while N17rac1 expression inhibited phenylephrine-induced leucine incorporation. These results suggest rac1 is an essential element of the signaling pathway leading to cardiac myocyte hypertrophy.