Induction of connective tissue growth factor (CTGF) in human endothelial cells by lysophosphatidic acid, sphingosine-1-phosphate, and platelets
Introduction
Atherosclerosis, from its early stages, is characterized by the activation of endothelial cells by a broad variety of atherogenic stimulants such as increased levels of lipids, hyperglycemia, or activated platelets. Thereby, endothelial cells loose their physiological role as a semi-permeable membrane, which separates the blood cells and soluble components, such as proteins or lipoprotein complexes, from the cells of the intima, primarily vascular smooth muscle cells. Loss of this functional barrier and endothelial dysfunction are considered early events in atherosclerosis. This stage in the development of atherosclerosis is nowadays regarded to be a complex process involving interactions between lipids and activated cells of the immune system, T cells, monocytes or macrophages, which then convert into foam cells. [1]. Endothelial dysfunction itself involves the release of many biological mediators, among them bioactive lipids such as sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) [2], [3].
The exact pro-atherogenic role of S1P is still under investigation. S1P is primarily found in the HDL fraction and in platelets, and was shown to mediate endothelial cell migration and survival [4]. Furthermore, S1P stimulates new blood vessel formation in combination with vascular endothelial growth factor or basic fibroblast growth factor in vivo and in vitro [5]. The pro-atherogenic role of LPA includes changes in the endothelial cell cytoskeleton and modulation of migration, cell–cell interaction, and proliferation. In general, the effects of LPA are more restricted to certain types of endothelial cells [5].
Connective tissue growth factor (CTGF) is another mediator with an emerging role in atherosclerosis. CTGF is widely recognized as a fibrogenic factor, but was originally described as a protein released from serum-stimulated endothelial cells [6]. Afterwards, CTGF was shown to be expressed in atherosclerotic lesions [7] and to mediate adherence and migration of monocytes, vascular smooth muscle cells, and activated platelets suggesting a functional role in the development of atherosclerotic lesions and plaque rupture [8], [9]. Endothelial cells themselves are targets of CTGF leading to proliferation, migration, and in vitro and in vivo angiogenesis [10], [11]. The capacity of CTGF to induce angiogenesis depends on the interaction with other angiogenic factors, because CTGF was also shown to bind VEGF and thus inhibit angiogenesis [12].
Expression of CTGF by endothelial cells is only partially characterized. Its regulation seems to vary depending on the type of endothelial cell investigated. Differences were observed between endothelial cells obtained from large vessels, where cAMP inhibited degradation of CTGF, and endothelial cells from small vessels, where cAMP stimulated CTGF protein degradation [13]. Contradictory results were obtained in terms of CTGF regulation by VEGF: whereas up-regulation of CTGF gene expression was reported in retinal capillary cells [14], no effect of VEGF was detected in transformed retinal endothelial cells [15].
The aim of the present study was to further investigate the regulation of CTGF in endothelial cells with special emphasis on the modulation of CTGF by the lipid mediators LPA and S1P. Primary human umbilical vein endothelial cells (HUVEC) and a HUVEC-derived cell line (EAHY 926) were used as model systems. Freshly isolated platelets, as a potential source of LPA and S1P, were also co-incubated with endothelial cells.
Section snippets
Materials
Cell culture reagents were from Gibco (Eggenstein, Germany), Promo Cell (Heidelberg, Germany) or Biochrom (Berlin, Germany). Accutase™ was from PAA Laboratories (Linz, Austria). Geranylgeranylpyrophosphate (GGPP), farnesylpyrophosphate (FPP), mevalonic acid lactone, PD98059, pertussis toxin, S1P and LPA were from Sigma (Munchen, Germany). GGPP and FPP were dissolved in methanol/10 mM NH4OH. Mevalonic acid lactone was converted to sodium mevalonate as described by Essig et al. [16]. Simvastatin
Induction of CTGF in EAHY 926 cells
Throughout this study, the HUVEC-derived cell line EAHY 926 and primary cultures of HUVEC were used in parallel. As a cell line, EAHY 926 cells were abundant and without biological variability due to individual donors, which had to be taken into consideration when working with primary cultures. Incubation of EAHY 926 cells with LPA (10 μM) or S1P (1 μM) led to a time-dependent increase in CTGF mRNA and protein expression (Fig. 1A). In human cells, CTGF may be N-glycosylated giving rise to two
Discussion
This study further elucidates the potential role of CTGF in atherogenesis. In our study we showed that LPA and S1P, constituents of lipoproteins and released by activated platelets, induce CTGF in endothelial cells, EAHY 926 and HUVEC. LPA was previously shown to induce CTGF in renal cells [22], [26], whereas thus far, S1P had not been characterized as an inducer of CTGF. In a very recent report, CTGF induction by S1P was confirmed in mesangial cells using a cDNA array approach [27]. Both, S1P
Acknowledgements
This work was supported by the Deutsche Forschungsgemeinschaft, Graduiertenkolleg 750. The expert technical assistance of M. Rehm and S. Kesting is gratefully acknowledged. We thank J. Heusinger-Ribeiro for providing us with the supernatants containing over-expressed CTGF. The authors appreciate the critical reading of the manuscript by I. Cicha.
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