quantification [22] and data were normalized to ribosomal 18S RNA expression. In vitro morphogenesis assay Matrigel (50 ml/well, 8–12 mg/ml) was pipetted into 13 mm tissue culture wells and polymerized for 30 min to 1 h at 37uC, as described [23]. Human mesoangioblasts were plated (206103/ well) in Mega Cell Medium, supplemented with 2% FCS in the presence or absence of 1 mmol/L S1P or 10 mmol/L SKI-2. The effects on the morphological changes of mesoangioblats were recorded after 24 h with an inverted microscope (Leitz DM_IRB) equipped with CCD optics and a digital analysis system. Statistical analysis Densitometric analysis of the Western Blot bands was performed using ImageJ and reported as percentage relative to the intensity of the band corresponding to control set as 100. Data are expressed as means 6 S.E.M. of at least three experiments. Statistical analysis was performed using Student’s t-test. Asterisks indicate statistical significance: *P,0.05. Results A cDNA microarray study was performed to establish transcriptional profiles of human mesoangioblasts treated with the sphingolipid. For this purpose, when human mesoangioblasts were 90% confluent, the medium was changed to DMEM containing 0.1% BSA and further incubated in the presence of 1 mmol/L S1P for 6 h or 24 h. In our set of four arrays 369 up-regulated and 166 down- regulated genes were found to be significant (Supplemental table S1,S2). As shown in Fig. 1, after analysis of the top 25 up-regulated and top 25 down-regulated genes by S1P we found that genes involved in apoptosis induction such as PMAIP1 and BBC3 were significantly down-regulated. PMAIP1 and BBC3 encode for Noxa and Puma respectively that belong to the BH3-only Bcl2 pro- apoptotic family of proteins [24]. On the contrary, but consistently with these results, the anti-apoptotic gene PPP1R13L, which encodes for a protein that blocks apoptosis by presumably binding and blocking p53, was found to be up-regulated following S1P challenge [25]. The S1P-dependent regulation of genes involved in apoptosis confirmed the anti-apoptotic role of this sphingolipid previously demonstrated in these progenitor cells [11]. Moreover, as shown in Fig. 1, S1P regulated the expression of genes involved in SM differentiation such as the transcription factors GATA6 and TRPS1 and those encoding for SM contractile proteins CNN1 and TPM1. GATA6 is the only member of the GATA family transcription factors that is expressed in VSMC [26] and is rapidly down- regulated upon mitogen stimulation [27]. Although the down- stream SM-specific genes targeted by GATA6 has yet to be completely identified, it plays a crucial role in the maintenance of the differentiated phenotype in VSMC [28]. Interestingly, in concert with S1P-induced up-regulation of GATA6 expression, TRPS1 which encodes for a nuclear protein that binds GATA consensus motif and potently represses transcriptional activation mediated by other GATA factors [29], was among the genes maximally down-regulated following S1P treatment. From the complete analysis of the microarray data (Supple- mental tables S1,S2) emerged that the gene encoding for the LIM domain protein, LMCD1, was significantly up-regulated after S1P treatment. Expression of LMCD1 is remarkably similar to that of GATA6, with high level of expression observed in vascular SM cells and myocardium [30]. S1P Differentiates Mesoangioblasts Notably, mRNA encoding for SM contractile apparatus proteins such as CNN1, that binds very strongly to actin in a Ca2+-independent manner, and the thin filament component TPM1 were among the most significantly up-regulated by S1P (Fig. 1). Furthermore, the expression of genes encoding for the SM-specific actin-binding protein caldesmon (CALD1) and TAGLN was also found to be significantly augmented (Supple- mental table S1) supporting the view that not only S1P controls the transcriptional machinery which gives rise to the SM phenotype but S1P itself is also responsible for the onset of the differentiated phenotype. From the analysis of the transcriptional profile, Necdin and Msx2, previously identified as transcription factors required for SM differentiation of mesoangioblasts [3,4], were not found to be modified by S1P treatment. Subsequently, in order to confirm the cDNA microarray findings, we performed a Real Time RT-PCR analysis of specific genes involved in SM differentiation that were differentially expressed following S1P challenge. Mesoangioblast treatment with 1 mmol/L S1P for 6 h and 24 h significantly increased mRNA levels of TPM1, GATA6, CNN1 and LMCD1, while it significantly decreased that of TRPS1. These results are consistent with that of the microarray data (Fig. 2A). The fold increase of TPM1 and GATA6 mRNA expression levels were higher at 6 h, while those of CNN1 and LMCD1 were higher at 24 h treatment with the sphingolipid, indicating a different time-course of expression for these genes. In agreement, GATA6 expression was also found more potently increased by S1P at 6 h than at 24 h in the cDNA microarray (Fig. 1). Total lysates of serum-starved mesoangioblasts challenged with 1 mmol/L S1P for 24 h and 48 h were then analyzed by Western blot analysis for SM contractile apparatus proteins CNN1 and TPM1, and the transcription factor GATA6. S1P increased protein levels of all investigated markers after 24 h of incubation and its effect was still significant at 48 h (Fig. 2B). The morphological change provoked by this bioactive sphingolipid was clearly appreciable by confocal immunofluorescent micro- scopic analysis of mesoangioblasts performed to reveal the state of assembly of the cytoskeleton. Indeed, cell incubation with 1 mmol/ L S1P for 48 h, profoundly altered the organization of actin cytoskeleton, giving rise to the formation of numerous F-actin filaments organized as stress fibers anchored to the plasma membrane, which were immunorevealed by TRITC-phalloidin (Fig. 2C). Notably, the same figure shows that a certain number of mesoangioblasts (approximately 30–40%) was strongly positive to the antibodies against CNN1 and TPM1 after S1P treatment. Thus, data presented in Figs. 2B and 2C demonstrate that the effect elicited by S1P on mesoangioblast protein levels was consistent with changes of the transcriptional profile induced by this sphingolipid. The involvement of S1PR in the differentiating action of S1P was next examined. Interestingly, as shown in Fig. 2D, pre- incubation of human mesoangioblasts with the specific antagonist of S1P1/3 VPC23019 (1 mmol/L) or the specific antagonist of S1P2 JTE013 (1 mmol/L) completely abolished the increase of the SM marker CNN1 induced by 1 mmol/L S1P, suggesting that S1P2 and S1P1/3 receptors mediate distinct molecular events necessary for the differentiation to occur. Given that GATA6 is recognized as a transcription factor critical for SM differentiation [31], to better characterize the increase of this transcription factor induced by S1P, we next determined if the sphingolipid augmented GATA6 mRNA expression levels in a dose-dependent manner. As shown in Fig. 3A, the sphingolipid was efficacious in upregulating GATA6 mRNA at a concentration of 100 nmol/L up to 1 mmol/L, while PLoS ONE | www.plosone.org 3 May 2011 | Volume 6 | Issue 5 | e20389