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Besides the well known pathophysiological impact of oxidative stress in cardiovascular disease, reactive oxygen species (ROS) generated at low concentrations exert a role as signaling molecules that are involved in signal transduction cascades of numerous growth factor-, cytokine-, and hormone-mediated pathways, and regulate biological effects such as apoptosis, cell proliferation, and differentiation. Embryonic stem cells have the capacity to differentiate into the cardiovascular cell lineage. Furthermore, upon confrontation culture with tumor tissue, they form blood vessel-like structures that induce tumor-induced angiogenesis within tumor tissues. The role of ROS in cardiovascular differentiation of embryonic stem cells appears to be antagonistic. Whereas continuous exposure to ROS results in inhibition of cardiomyogenesis and vasculogenesis, pulsechase exposure to low-level ROS enhances differentiation toward the cardiomyogenic as well as vascular cell lineage. This review summarizes the current knowledge of ROS-induced cardiovascular differentiation of embryonic stem cells as well as the role of ROS in tumor-induced angiogenesis.Antioxid. Redox Signal. 7, 1423–1434.

Platelet-derived growth factor BB (PDGF-BB) has been assigned a critical role in vascular growth and recruitment of perivascular mural cells. The purpose of the present study is to investigate the signalling events underlying the stimulation of vasculogenesis of mouse embryonic stem (ES) cells by PDGF-BB. PDGF-BB increased vascular sprouting and branching of capillary-like structures in embryoid bodies as evaluated by computer-assisted analysis of CD31-positive cell structures. It also activated extracellular-regulated kinase 1,2 (ERK1,2) and c-Jun N-terminal kinase but not p38 mitogen-activated protein kinase or PI 3-kinase. Microfluorometric analysis of fluo-4 fluorescence revealed that treatment with PDGF-BB raised intracellular Ca²⁺ levels in differentiating ES cells expressing the PDGF receptor β, an effect that was abolished in the presence of the intracellular Ca²⁺ chelator BAPTA. Furthermore, PDGF-BB raised reactive oxygen species (ROS) levels in embryoid bodies as evaluated using the redox-sensitive dye H₂DCF-DA. ROS generation was blunted in the presence of the NADPH oxidase inhibitors diphenylen iodonium (DPI) and apocynin as well as in the presence of BAPTA, suggesting that ROS generation is regulated by intracellular Ca²⁺ transients. The stimulation of vasculogenesis of ES cells upon treatment with PDGF-BB was significantly inhibited by the ERK1,2 inhibitor U0126, the NADPH oxidase inhibitors DPI, apocynin, 4-(2-aminoethyl)benzenesulfonylfluoride and VAS2870, the free radical scavengers vitamin E, and N-(2-mercaptopropionyl)glycin as well as by BAPTA. Our data demonstrate that the pro-vasculogenic effects of PDGF-BB are mediated by Ca²⁺-induced ROS generation, resulting in the activation of an ERK1,2-mediated signal transduction cascade.

Hyperthermia is an important component of many cancer treatment protocols. In our study the regulation of the multidrug resistance (MDR) transporter P‐glycoprotein by hyperthermia was studied in multicellular prostate tumor spheroids. Hyperthermia treatment of small (50–100 μm) tumor spheroids significantly increased P‐glycoprotein and mdr‐1 mRNA expression with a maximum effect at 42°C, whereas only moderate elevation of P‐glycoprotein was found in large (350–450 μm) tumor spheroids. Hyperthermia caused an elevation of intracellular reactive oxygen species (ROS). Inhibition of ROS generation with NADPH‐oxidase inhibitors diphenylen iodonium (DPI) and 4‐(2‐aminoethyl)benzenesulfonyl fluoride (AEBSF) abolished P‐glycoprotein expression but did not affect its transcript levels following heat treatment. This indicates that P‐glycoprotein levels are controlled by regulating its translation rate or stability. Hyperthermia incubation resulted in a differential activation of p38 mitogen‐activated protein kinase (MAPK), extracellular regulated kinase 1,2 (ERK1,2), and c‐jun N‐terminal kinase (JNK) immediately, 4 hr and 24 hr after treatment. Furthermore, upregulation of hypoxia‐inducible factor 1α (HIF‐1α) was observed. Elevation of HIF‐1α and P‐glycoprotein expression following hyperthermia treatment were abolished upon coadministration of the p38 inhibitor SB203580. In contrast the JNK inhibitor SP600125 and the ERK1,2 inhibitor UO126 resulted in increase of HIF‐1α and P‐glycoprotein in the control as well as the hyperthermia‐treated samples, indicating negative regulation of intrinsic HIF‐1α and P‐glycoprotein expression by ERK1,2 and JNK signaling cascades. In summary our data demonstrate that hyperthermia‐induced upregulation of P‐glycoprotein and HIF‐1α is mediated by activation of p38, whereas ERK1,2 and JNK are involved in repression of P‐glycoprotein and HIF‐1α under control conditions.

Peroxisome proliferator-activated receptors (PPARα, -β and -γ) are nuclear receptors involved in transcriptional regulation of lipid and energy metabolism. Since the energy demand increases when cardiac progenitor cells are developing rhythmic contractile activity, PPAR activation may play a critical role during cardiomyogenesis of embryonic stem (ES) cells. It is shown that ES cells express PPARα, -β, and -γ mRNA during differentiation of ES cells towards cardiac cells. Treatment with PPARα agonists (WY14643, GW7647, and ciprofibrate) significantly increased cardiomyogenesis and expression of the cardiac genes MLC2a, ANP, MHC-β, MLC2v, and cardiac α-actin. Furthermore, WY14643 increased PPARα gene expression and the expression of the cardiogenic transcription factors GATA-4, Nkx2.5, DTEF-1, and MEF 2C. In contrast, the PPARα antagonist MK886 decreased cardiomyogenesis, whereas the PPARβ agonist L-165,041 as well as the PPARγ agonist GW1929 were without effects. Treatment with PPARα, but not PPARβ, and PPARγ agonists and MK886, resulted in generation of reactive oxygen species (ROS), which was inhibited in the presence of the NADPH oxidase inhibitors diphenylen iodonium (DPI) and apocynin and the free radical scavengers vitamin E and N-(2-mercapto-propionyl)-glycine (NMPG), whereas the mitochondrial complex I inhibitor rotenone was without effects. The effect of PPARα agonists on cardiomyogenesis of ES cells was abolished upon preincubation with free radical scavengers and NADPH oxidase inhibitors, indicating involvement of ROS in PPARα, mediated cardiac differentiation. In summary, our data indicate that stimulation of PPARα but not PPARβ and -γ enhances cardiomyogenesis in ES cells using a pathway that involves ROS and NADPH oxidase activity. Disclosure of potential conflicts of interest is found at the end of this article.