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,1,
* Department of Hemostasis and Transfusion Medicine, and
Center for Biological and Medical Research, and
Institute of Molecular Medicine, Heinrich-Heine University, Düsseldorf, Germany
1Correspondence: Department of Hemostasis and Transfusion Medicine, Laboratory of Vascular Biology, Bldg. 12.49.00, Rm. 9, Moorenstr. 5, D-40225 Düsseldorf, Germany. E-mail: stoldt{at}uni-duesseldorf.de
SPECIFIC AIMS
In this study, we investigated how TGF-ßbeta;1 and cell cycle-specific events can regulate prothrombotic and antithrombotic protein expression and modulate the hemostatic microenvironment in endothelial cells. Our findings demonstrate that the expression of a subset of hemostatically relevant proteins is regulated during endothelial cell cycle and that TGF-ßbeta;1 can differentially modulate cell cycle-controlled protein expression.
PRINCIPAL FINDINGS
1. Prothrombotic and antithrombotic molecule expression during the cell cycle
To investigate cell cycle-regulated protein expression, we characterized human umbilical vein endothelial cells (HUVEC) in the G1 and in the S/G2 phase by DNA staining with propidium iodide. A secondary immunostaining with specific antibodies revealed that thrombomodulin (Fig. 1
), both plasminogen activators (u-PA and t-PA), and plasminogen activator inhibitor 1 (PAI-1) were predominantly expressed in the S/G2 phase compared to the G1 phase. As assessed by flow cytometric analyses, the expression of thrombomodulin, t-PA and u-PA increased by 31%, 41%, and 41%, respectively, in replicating tetraploid cells, compared to G1-arrested diploid cells. The cell cycle-dependent expression of PAI-1 increased even by up to 72% in the S/G2 phase compared to cells in the G1-phase, whereas the expression of von Willebrand factor (vWF) remained unaltered during progression of the cell cycle.
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2. Prothrombotic and antithrombotic molecule expression on TGF-ßbeta;1 treatment
TGF-ßbeta;1 arrests endothelial cells mainly in the G1 phase. When cells have passed the G1/S transition, they are not longer responsive to TGF-ßbeta;1-dependent cell cycle control. Thus, after incubation with TGF-ßbeta;1, the expression of hemostatically relevant molecules in the G1 and S/G2 phase is comparable. On treatment with TGF-ßbeta;1, the cell cycle-dependent expression of thrombomodulin was elevated by 
34% in the S/G2 phase as compared to the G1 phase. Expression of t-PA and u-PA showed an increase of 66% and 49%, respectively, when replicating tetraploid cells were treated with 1 ng/ml TGF-ßbeta;1 in comparison to the equally treated diploid cells. When the combined effects of TGF-ßbeta;1 and cell cycle-dependent protein expression were studied, an increase of PAI-1 expression by 165% was observed. In contrast, no change in the expression concentration of vWF was observed under the same experimental conditions.
3. Specific induction of PAI-1 expression by TGF-ßbeta;1 in the S/G2 phase
To investigate the potential physiological relevance of the S/G2 phase and the role of TGF-ßbeta;1 in modulating the expression of prothrombotic and antithrombotic molecules in growing HUVEC, we compared their expression during the cell cycle in the presence and absence of TGF-ßbeta;1. The ratio of cells expressing prothrombotic and antithrombotic molecules in the S/G2 phase of TGF-ßbeta;1-treated and untreated HUVEC represents the hemostatic capacity during endothelial growth. Figure 2
demonstrates that cells in the S/G2 phase augment PAI-1 expression on treatment with 1 ng/ml of TGF-ßbeta;1. The protein expression ratio (G2/G1) increased from 1.7 to 2.9. In contrast, this ratio was not affected by TGF-ßbeta;1 with regard to the expression of thrombomodulin, t-PA, and u-PA. This finding demonstrates that TGF-ßbeta;1 specifically modulates cell cycle-controlled expression of PAI-1.
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CONCLUSIONS AND SIGNIFICANCE
It is well established that TGF-ßbeta;1 has the ability to arrest cells in the G1 phase and to regulate the expression of several proteins involved in endothelial homeostasis. However, whether and how both endothelial responses to TGF-ßbeta;1 are linked to each other, is unknown. Our findings now constitute the first report demonstrating that growing endothelial cells modulate their hemostatic microenvironment in a cell cycle-dependent manner under the specific control of TGF-ßbeta;1.
Resting endothelial cells are hemostatically balanced (Fig. 3
). In contrast, growing endothelium in the S/G2 phase increases the expression of antithrombotic molecules, including thrombomodulin, u-PA, and t-PA, and the prothrombotic molecule PAI-1. Interestingly, vWF is constantly expressed throughout the cell cycle. Thus, not all hemostatically relevant proteins are under cell cycle control, and the potential to influence the hemostatic properties of the endothelium in a cell cycle-dependent manner is restricted to a specific subset of proteins.
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The expression of thrombomodulin, t-PA, and u-PA increase in the S/G2 phase compared to the G1 phase of TGF-ßbeta;1-treated cells. On treatment with TGF-ßbeta;1, expression of PAI-1 show an extensive net increase in the S/G2 phase. Thus, growing endothelial cells produce an antifibrinolytic and prothrombotic microenvironment in a cell cycle-dependent manner and under specific control of TGF-ßbeta;1 (Fig. 3)
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In summary, the enhanced expression of PAI-1 that is triggered by TGF-ßbeta;1 selectively in the S/G2 phase might constitute a new aspect of hemostatic functions of endothelial cells, which could be relevant for different conditions, including angiogenesis and wound healing.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3032fje
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