Jeanette A.M. Maier

In vitro inhibition of endothelial cell growth by the antiangiogenic drug AGM-1470 (TNP-470) and the anti-endoglin antibody TEC-11

Angiogenesis plays a key role in tumor growth, progression and metastasis. The modulation of angiogenesis represents a potentially useful target for novel forms of anticancer therapy. Two such modulators are AGM-1470 (TNP-470, angioinhibin), which is a synthetic analog of the antibiotic fumagallin, and the monoclonal antibody TEC-11 to endoglin. We investigated the mechanisms of action of these modulators on human microvascular and macrovascular endothelial cells and on the transformed endothelial cell line ECV-304 in vitro. The administration of AGM-1470 or TEC-11 resulted in a significant inhibition of cell proliferation in all cell types used; this effect was reversible upon removal of these compounds from the culture medium. Furthermore, biochemical and morphological analyses showed that neither AGM-1470 or TEC-11 induce apoptosis. Both AGM-1470 and TEC-11 inhibited the production of urokinase-type plasminogen activator (u-PA), an enzyme involved in the early steps of neovascularization. Finally, the incubation of endothelial cells with both AGM-1470 and TEC-11 did not produce an additive effect on growth cell inhibition, apoptosis or u-PA production. Since both AGM 1470 and TEC-11 inhibit crucial events such as endothelial cell growth and protease production, our results provide a basis for their therapeutic use as angiostatic molecules in cancer.

EDF-1, a Novel Gene Product Down-regulated in Human Endothelial Cell Differentiation

Endothelial cell differentiation is a crucial step in angiogenesis. Here we report the identification of EDF-1, a novel gene product that is down-regulated when endothelial cells are induced to differentiate in vitro. The cDNA encodingEDF-1 was isolated by RNA fingerprinting from human endothelial cells exposed to human immunodeficiency virus type 1 Tat, a viral protein known to be angiogenic. The deduced amino acid sequence of EDF-1 encodes a basic intracellular protein of 148 amino acids that is homologous to MBF1 (multiprotein-bridgingfactor 1) of the silkworm Bombyx mori and to H7, which is implicated in the early developmental events of Dictyostelium discoideum. Interestingly, human immunodeficiency virus type 1 Tat, which affects endothelial functions, and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate and culture on fibrin gels, which promote endothelial differentiationin vitro, all down-regulate EDF-1 expression both at the RNA and protein levels. In addition, the inhibition of EDF-1 translation by an antisense anti-EDF-1 construct results in the inhibition of endothelial cell growth and in the transition from a nonpolar cobblestone phenotype to a polar fibroblast-like phenotype. These data suggest that EDF-1 may play a role in the regulation of human endothelial cell differentiation.

Induction of human endothelial cell growth by mildly oxidized low density lipoprotein.

The aim of the present study was to examine whether endothelial growth could be modulated by mildly oxidized low density lipoprotein. When human endothelial cells were cultured in the presence of mildly oxidized low density lipoprotein (1 microgram/ml), a significant induction of endothelial cell growth was observed, whereas native low or high density lipoprotein were ineffective. Further, treatment of endothelial cells with mildly oxidized low density lipoprotein modulated the expression of cytokines and growth factors which may be relevant in atherogenesis. Endothelial cells chronically exposed to mildly oxidized low density lipoprotein underwent a more rapid onset of cellular senescence. Since senescence is associated with endothelial dysfunction, the novel finding showing that mildly oxidized low density lipoprotein induces endothelial cell growth may be relevant in the development and evolution of the atherosclerotic lesions.

Serum From Magnesium-Deficient Rats Affects Vascular Endothelial Cells in Culture: Role of Hyperlipemia and Inflammation

Abstract Magnesium (Mg) deficit has been implicated as a risk for atherosclerosis. An important characteristic of experimental Mg deficiency in rats is the spontaneous inflammation with marked leukocytosis and increase in plasma concentrations of inflammatory cytokines. This deficiency is also accompanied by hyperlipemia resulting from accumulation of triglyceride-rich lipoproteins (TGRLP). The present investigation was performed to determine the effect of serum from Mg-deficient animals on cultured vascular endothelial cells. Sera were obtained from control and Mg-deficient rats fed for 8 days adequate or Mg-deficient diets. Mg-deficient animals presented an important leukocytosis and an increased interleukin (IL) 6 concentration in the plasma. Deficient rats were hypertriglyceridemic as compared with control ones, but their cholesterolemia was not modified significantly. Pooled sera from control and Mg-deficient animals were added to the culture medium of human umbilical endothelial cells (HUVEC). The results show that serum from Mg-deficient rats stimulates proliferation of cultured endothelial cells, increases adhesion of monocytes to these cells, and causes an induction of plasminogen activator inhibitor factor 1 (PAI-1) mRNA level in these cells. The present study demonstrates that the inflammatory and hyperlipemic serum from Mg-deficient animals affects various processes in endothelial cells, which are known to be implicated in atherogenesis.

Anti-tumor immunity induced by murine melanoma cells transduced with the Mycobacterium tuberculosis gene encoding the 38-kDa antigen

The Mycobacterium tuberculosis Ag38 gene, which encodes a highly immunogenic protein, was cloned into a retroviral vector in-frame with the leader and the transmembrane portion of the nerve growth factor receptor, and transduced into murine melanoma cell line B16-B78. Significant protection was observed in mice immunized with the transduced melanoma cells and subcutaneously challenged with parental melanoma cells since only 20% of mice developed tumors. Necroscopy of mice immunized with the transduced melanoma cells revealed dramatic inhibition of experimental metastases induced by intravenous (i.v.) inoculation of parental melanoma cells. Moreover, vaccination with transduced cells significantly prolonged survival of mice challenged i.v. with parental melanoma cells. These data indicate that the presence of the mycobacterial 38-kDa protein greatly enhances immunological recognition of structures expressed by the parental melanoma cells. Comparison of Th1 and Th2 responses in mice immunized with parental melanoma cells versus mice receiving the transduced cells revealed a clear predominance of Th1 responses when the Ag38 protein was endogenously expressed. This transduction approach may represent a promising immunotherapeutic strategy for the treatment of cancer patients.