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Complete transcriptome profiling of contractile smooth muscle cells (SMCs) differentiated from embryonic stem cells is crucial for the characterization of smooth muscle gene expression signatures and will contribute to defining biological and physiological processes in these cells. We have generated a transgenic embryonic stem cell line expressing both the puromycin acetyl transferase and enhanced green fluorescent protein cassettes under the control of the Acta2 promoter. Applying a specific monolayer culture protocol using retinoic acid, a puromycin-resistant and enhanced green fluorescent protein-positive Acta2(+) SMC population of 95% purity was isolated. Acta2(+) SMCs were characterized by semiquantitative and quantitative RT-PCR profiling of SMC markers and by microarray expression profiling, as well as by immunostaining for SMC-specific cytoskeletal proteins. Patch-clamp electrophysiological characterization of these cells identified SMC-specific channels such as the ATP-sensitive potassium channel and the Ca(2+)-activated potassium channel. Culturing of Acta2(+) SMCs in serum-containing medium resulted in a significant number of hypertrophic and binucleated cells failing to complete cell division. Functional characterization of the cells has been proved by stimulation of the cells with vasoactive agents, such as angiotensin II and endothelin. We concluded that our embryonic stem cell-derived SMC population possesses the contractile and hypertrophic phenotype of SMCs incapable of proliferation. This is the first study describing the complete transcriptome of ES-derived SMCs allowing identification of specific biological and physiological processes in the contractile phenotype SMCs and will contribute to the understanding of these processes in early SMCs derived from embryonic stem cells. [ABSTRACT FROM AUTHOR]

Recently, growing attention has been directed toward stem cell metabolism, with the key observation that the plasticity of stem cells also reflects the plasticity of their energy substrate metabolism. There seems to be a clear link between the self-renewal state of stem cells, in which cells proliferate without differentiation, and the activity of specific metabolic pathways. Differentiation is accompanied by a shift from anaerobic glycolysis to mitochondrial respiration. This metabolic switch of differentiating stem cells is required to cover the energy demands of the different organ-specific cell types. Among other metabolic signatures, amino acid and carbohydrate metabolism is most prominent in undifferentiated embryonic stem cells, whereas the fatty acid metabolic signature is unique in cardiomyocytes derived from embryonic stem cells. Identifying the specific metabolic pathways involved in pluripotency and differentiation is critical for further progress in the field of developmental biology and regenerative medicine. The recently generated knowledge on metabolic key processes may help to generate mature stem cell-derived somatic cells for therapeutic applications without the requirement of genetic manipulation. In the present review, the literature about metabolic features of stem cells and their cardiovascular cell derivatives as well as the specific metabolic gene signatures differentiating between stem and differentiated cells are summarized and discussed.

Objective: We recently isolated and characterized endothelial-like CD31(+) cells derived from mouse embryonic stem (mES) cells and identified their transcriptome. The main objective of this study was to determine the functional relevance of the transcripts of unknown function (TUF) for vasculature development., Methods and Results: We selected 2 TUFs of more than 27 to study their role for blood vessel development in zebrafish. Morpholino (MO) knockdown of the zebrafish orthologs of the first TUF (TUF1, mouse cDNA BC022623) showed disruption of the intersegmental vessels (ISV) at 2 days postfertilization as observed by live imaging of fli:EGFP-transgenic embryos. The morphants showed abnormal blood circulation, but no effect on hematopoiesis was observed as demonstrated by gata-1 in situ hybridizations. Because knockdown of TUF1 resulted in disruption of the ISV patterning we named the TUF1 somitovasculin. TUF2 has been identified as cDNA clone BC020535. The MO knockdown of TUF2 resulted in a phenotype with an enlarged heart and the embryos lacked circulation completely., Conclusions: We have shown the participation of a novel transcript (named somitovasculin) in circulatory vessel development. The combination of expression profiling in differentiating mES cells and the zebrafish model has the potential for rapid identification and functional characterization of TUFs.

Recent evidence indicates that epigallocatechin 3-O-gallate (EGCG), the major catechin derived from green tea leaves, lowers the risk of cardiovascular diseases such as atherosclerosis and hypertension. However, a precise mechanism for this biologic function has not yet been clearly delineated. Angiotensin II (Ang II) stimulates vascular smooth muscle cell (VSMC) hypertrophy, which is a critical event in the development of atherosclerosis, hypertension, and angioplasty-induced restenosis. In the present study, we show that EGCG inhibits Ang II-stimulated VSMC hypertrophy, as determined by [3H]leucine incorporation into VSMC. Since mitogen-activated protein kinase (MAPK) families are involved in cell growth, we determined whether EGCG affects them. EGCG pretreatment did not exert any significant changes in Ang II-stimulated activation of extracellular signal-regulated kinase (ERK) and p38 MAPK. EGCG only inhibited Ang II-stimulated activation of c-Jun N-terminal kinase (JNK). Moreover, EGCG suppressed Ang II-induced c-jun mRNA expression. In contrast, EGC, a structural analogue of EGCG, did not inhibit the JNK activity or c-jun mRNA expression. In addition, a specific JNK inhibitor, SP600125, dose-dependently suppressed Ang II-stimulated VSMC hypertrophy. These results suggest that the effect of EGCG on Ang II-induced VSMC hypertrophy is due to specific inhibition of the JNK signaling pathway at both transcriptional and posttranslational levels, which may underlie its beneficial effect on the cardiovascular diseases.

AT(1) receptor activation leads to vasoconstriction, blood pressure increase, free radical release, and cell growth. AT(1) receptor regulation contributes to the adaptation of the renin-angiotensin system to long-term stimulation and serves as explanation for the involvement of the AT(1) receptor in the pathogenesis of cardiovascular disease. The molecular mechanisms involved in AT(1) receptor regulation are poorly understood. Here, we report that angiotensin II accelerates AT(1) receptor mRNA decay in vascular smooth muscle cells. A cognate mRNA region within the 3' untranslated region at bases 2175 to 2195 governs the inducible decay of the AT(1) receptor mRNA. Sequential protein purifications led to the discovery of a novel mRNA binding protein, calreticulin, which mediates destabilization of the AT(1) receptor mRNA. Angiotensin II-caused phosphorylation of calreticulin enables binding of calreticulin to the AT(1) receptor mRNA at bases 2175 to 2195 and propagates calreticulin-induced acceleration of AT(1) receptor mRNA decay. Thus, a novel mRNA binding protein, calreticulin, is discovered, which causes AT(1) receptor mRNA degradation via binding to a distinct mRNA region in the 3' untranslated region. These findings display a novel mechanism of posttranscriptional mRNA processing.

We recently demonstrated that the mitogenic effect of LDL (100 microg/mL) as well as its early intracellular signaling pathway are mediated by a pertussis-toxin (PTX)-sensitive G(i) protein-coupled receptor that is independent from its classical receptor and involves activation of extracellular response kinases (ERK1/2) (also known as p44(mapk)/p42(mapk)). In the present study we examined whether LDL-adherent factors may be responsible for some of the effects of LDL. The term "signaling activity" is used to characterize fractions that cause an increase in intracellular free Ca(2+) concentration or stimulate ERK1/2 and c-fos mRNA expression. LDL, HDL, and VLDL stimulate ERK1/2 with the following order of potency: LDL>HDL>VLDL. After delipidation of LDL with chloroform/methanol/water mixtures a PTX-sensitive signaling activity was found in one fraction arbitrarily called LDL-F. After further analysis of LDL-F compounds by high pressure liquid chromatography, a PTX-sensitive signaling activity was detected only in the fraction with a retention time of 33 minutes (arbitrarily called LDL-F33). Similarly, after separation of sphingosine-1-phosphate (SPP) and sphingosylphosphorylcholine (SPC) by high pressure liquid chromatography, a PTX-sensitive signaling activity was found in the fractions 33 and 33 to 35, respectively. These findings demonstrate that the effects of LDL-F33 are mimicked by similar fractions collected from SPP/SPC, hence suggesting that these LDL-adherent molecules are possibly closely related to SPP/SPC. A PTX-sensitive signaling activity was also detected in HDL and HDL-F33. Therefore, LDL and other lipoproteins may function as carriers for bioactive phospholipids thereby contributing to the development of coronary artery disease. Our findings support a new research concept that may contribute in elucidating cellular mechanisms promoting coronary artery disease.

Background: Because LDL and the angiotensin II type 1 (AT1) receptor are thought to be involved in the pathogenesis of chronic vascular disease, we studied possible interactions between these two biological systems in cultured rat vascular smooth muscle cells., Methods and Results: Incubation of vascular smooth muscle cells with 100 micrograms/mL LDL profoundly increased AT1 receptor mRNA to approximately 250% of control levels as assessed by Northern hybridization analysis. This effect is maximal 12 hours after addition of LDL to the culture medium and is sustained for up to 24 hours. The LDL-induced upregulation is dose dependent, with a maximal effect obtained with 100 micrograms/mL LDL. There is a correlative increase of cell surface-associated AT1 receptors as assessed by saturation radioligand binding assays. The half-life of AT1 receptor mRNA is increased substantially by LDL compared with that of cells treated only with 5,6-dichlorobenzimidazole to block transcription. Angiotensin II-induced elevation of cytosolic calcium concentration is significantly increased in vascular smooth muscle cells pretreated with LDL to 368 +/- 41 nmol/L compared with control cells pretreated with vehicle (248 +/- 33 nmol/L). Moreover, angiotensin II-induced DNA synthesis is markedly enhanced when cells are coincubated with 100 micrograms/mL LDL., Conclusions: These data reveal a significant upregulation of AT1 receptor gene expression by LDL in vascular smooth muscle cells through mechanisms that involve posttranscriptional mRNA stabilization. Ultimately, this AT1 receptor upregulation leads to an elevated functional response of vascular smooth muscle cells on angiotensin II stimulation.