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Retinoic acid (RA) facilitates tissue morphogenesis by regulating matrix matalloproteinase (MMPs) expression. Our objective was to examine the influence of RA on in vitro development of follicles enclosed within domestic cat ovarian tissues. Ovarian cortices from 9 prepubertal and 13 adult cats were incubated for 7 d in medium containing 0 (control), 1 or 5 μM RA and then analyzed for viability. Cortices from additional three animals of each age group were cultured in the same condition and follicle morphology, stage and size were histologically evaluated. In a separate study, cortices from 14 donors (7 prepubertal; 7 adult cats) were incubated in 0 or 5 μM RA for 7 d and assessed for (1) MMP1, 2, 3, 7, 9 and TIMP1 expression by qPCR and (2) protein expression of MMP9 by immunohistochemistry. Donor age did not influence follicle response to RA. Collective data from both age groups revealed that percentages of primordial follicles in 5 μM RA treatment were lower (P < 0.05; 40.5 ± 4.5%) than in fresh cortices (66.7 ± 5.3%) or controls (60.1 ± 4.0%) with 1 μM-RA treatment producing intermediate (56.3 ± 4.0%) results. Proportion of primary follicles in 5 μM RA (21.7 ± 3.3%) was higher than in fresh cortices (4.9 ± 2.9%) and controls (9.0 ± 2.8%) with 1 μM-RA treatment producing an intermediate value (13.8 ± 2.0%). Furthermore, proportion of secondary follicles increased after 7 d in the presence of 5 μM RA (9.5 ± 2.7%) compared to other groups (fresh, 1.9 ± 0.8%; control, 2.6 ± 1.1%; 1 μM RA, 2.5 ± 0.2%). MMP9 transcript and protein were upregulated, whereas MMP7 mRNA was suppressed by 5 μM-RA treatment compared to fresh counterparts. RA did not impact MMP1, 2, 3, 13 or TIMP1 expression. In summary, RA activated cat primordial follicle growth likely via a mechanism related to upregulation of MMP9 and down-regulation of MMP7 transcripts., Competing Interests: The authors have declared that no competing interests exist.

Although studies of the differentiation from mouse embryonic stem (ES) cells to vascular endothelial cells (ECs) provide an excellent model for investigating the molecular mechanisms underlying vascular development, temporal dynamics of gene expression and chromatin modifications have not been well studied. Herein, using transcriptomic and epigenomic analyses based on H3K4me3 and H3K27me3 modifications at a genome-wide scale, we analysed the EC differentiation steps from ES cells and crucial epigenetic modifications unique to ECs. We determined that Gata2, Fli1, Sox7 and Sox18 are master regulators of EC that are induced following expression of the haemangioblast commitment pioneer factor, Etv2. These master regulator gene loci were repressed by H3K27me3 throughout the mesoderm period but rapidly transitioned to histone modification switching from H3K27me3 to H3K4me3 after treatment with vascular endothelial growth factor. SiRNA knockdown experiments indicated that these regulators are indispensable not only for proper EC differentiation but also for blocking the commitment to other closely aligned lineages. Collectively, our detailed epigenetic analysis may provide an advanced model for understanding temporal regulation of chromatin signatures and resulting gene expression profiles during EC commitment. These studies may inform the future development of methods to stimulate the vascular endothelium for regenerative medicine., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The blood-brain barrier (BBB) is composed of four cell populations, brain endothelial cells (BECs), pericytes, neurons, and astrocytes. Its role is to precisely regulate the microenvironment of the brain through selective substance crossing. Here we generated an in vitro model of the BBB by differentiating human induced pluripotent stem cells (hiPSCs) into all four populations. When the four hiPSC-derived populations were co-cultured, endothelial cells (ECs) were endowed with features consistent with BECs, including a high expression of nutrient transporters (CAT3, MFSD2A) and efflux transporters (ABCA1, BCRP, PGP, MRP5), and strong barrier function based on tight junctions. Neuron-derived Dll1, which activates Notch signaling in ECs, was essential for the BEC specification. We performed in vitro BBB permeability tests and assessed ten clinical drugs by nanoLC-MS/MS, finding a good correlation with the BBB permeability reported in previous cases. This technology should be useful for research on human BBB physiology, pathology, and drug development., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Blood vessels are essential components for many tissues and organs. Thus, efficient induction of endothelial cells (ECs) from human pluripotent stem cells is a key method for generating higher tissue structures entirely from stem cells. We previously established an EC differentiation system with mouse pluripotent stem cells to show that vascular endothelial growth factor (VEGF) is essential to induce ECs and that cyclic adenosine monophosphate (cAMP) synergistically enhances VEGF effects. Here we report an efficient and robust EC differentiation method from human pluripotent stem cell lines based on a 2D monolayer, serum-free culture. We controlled the direction of differentiation from mesoderm to ECs using stage-specific stimulation with VEGF and cAMP combined with the elimination of non-responder cells at early EC stage. This "stimulation-elimination" method robustly achieved very high efficiency (>99%) and yield (>10 ECs from 1 hiPSC input) of EC differentiation, with no purification of ECs after differentiation. We believe this method will be a valuable technological basis broadly for regenerative medicine and 3D tissue engineering.

Specific neuronal types derived from embryonic stem cells (ESCs) can facilitate mechanistic studies and potentially aid in regenerative medicine. Existing induction methods, however, mostly rely on the effects of the combined action of multiple added growth factors, which generally tend to result in mixed populations of neurons. Here, we report that overexpression of specific transcription factors (TFs) in ESCs can rather guide the differentiation of ESCs towards specific neuron lineages. Analysis of data on gene expression changes 2 d after induction of each of 185 TFs implicated candidate TFs for further ESC differentiation studies. Induction of 23 TFs (out of 49 TFs tested) for 6 d facilitated neural differentiation of ESCs as inferred from increased proportion of cells with neural progenitor marker PSA-NCAM. We identified early activation of the Notch signaling pathway as a common feature of most potent inducers of neural differentiation. The majority of neuron-like cells generated by induction of Ascl1, Smad7, Nr2f1, Dlx2, Dlx4, Nr2f2, Barhl2, and Lhx1 were GABA-positive and expressed other markers of GABAergic neurons. In the same way, we identified Lmx1a and Nr4a2 as inducers for neurons bearing dopaminergic markers and Isl1, Fezf2, and St18 for cholinergic motor neurons. A time-course experiment with induction of Ascl1 showed early upregulation of most neural-specific messenger RNA (mRNA) and microRNAs (miRNAs). Sets of Ascl1-induced mRNAs and miRNAs were enriched in Ascl1 targets. In further studies, enrichment of cells obtained with the induction of Ascl1, Smad7, and Nr2f1 using microbeads resulted in essentially pure population of neuron-like cells with expression profiles similar to neural tissues and expressed markers of GABAergic neurons. In summary, this study indicates that induction of transcription factors is a promising approach to generate cultures that show the transcription profiles characteristic of specific neural cell types.

Mouse embryonic stem cells (ESCs) can differentiate into a wide range - and possibly all cell types in vitro, and thus provide an ideal platform to study systematically the action of transcription factors (TFs) in cell differentiation. Previously, we have generated and analyzed 137 TF-inducible mouse ESC lines. As an extension of this "NIA Mouse ESC Bank," we generated and characterized 48 additional mouse ESC lines, in which single TFs in each line could be induced in a doxycycline-controllable manner. Together, with the previous ESC lines, the bank now comprises 185 TF-manipulable ESC lines (>10% of all mouse TFs). Global gene expression (transcriptome) profiling revealed that the induction of individual TFs in mouse ESCs for 48 hours shifts their transcriptomes toward specific differentiation fates (e.g., neural lineages by Myt1 Isl1, and St18; mesodermal lineages by Pitx1, Pitx2, Barhl2, and Lmx1a; white blood cells by Myb, Etv2, and Tbx6, and ovary by Pitx1, Pitx2, and Dmrtc2). These data also provide and lists of inferred target genes of each TF and possible functions of these TFs. The results demonstrate the utility of mouse ESC lines and their transcriptome data for understanding the mechanism of cell differentiation and the function of TFs.

Poor engraftment of cells after transplantation to the heart is a common and unresolved problem in the cardiac cell therapies. We previously generated cardiovascular cell sheets entirely from pluripotent stem cells with cardiomyocytes, endothelial cells and vascular mural cells. Though sheet transplantation showed a better engraftment and improved cardiac function after myocardial infarction, stacking limitation (up to 3 sheets) by hypoxia hampered larger structure formation and long-term survival of the grafts. Here we report an efficient method to overcome the stacking limitation. Insertion of gelatin hydrogel microspheres (GHMs) between each cardiovascular cell sheet broke the viable limitation via appropriate spacing and fluid impregnation with GHMs. Fifteen sheets with GHMs (15-GHM construct; >1 mm thickness) were stacked within several hours and viable after 1 week in vitro. Transplantation of 5-GHM constructs (≈2 × 10(6) of total cells) to a rat myocardial infarction model showed rapid and sustained functional improvements. The grafts were efficiently engrafted as multiple layered cardiovascular cells accompanied by functional capillary networks. Large engrafted cardiac tissues (0.8 mm thickness with 40 cell layers) successfully survived 3 months after TX. We developed an efficient method to generate thicker viable tissue structures and achieve long-term survival of the cell graft to the heart.

Unlabelled: Opioid systems mainly regulate physiological functions such as pain, emotional tone and reward circuitry in neural tissues (brain and spinal cord). These systems are also found in extraneural tissues (ganglia, gut, spleen, stomach, lung, pancreas, liver, heart, blood and blood vessels), and recent studies have elucidated their roles in various organs. The current review focuses on the roles of opioid systems in blood vessels, especially angiogenesis, during development and tumour malignancy. The balance between endogenous activators and inhibitors of angiogenesis delicately maintains a normally quiescent vasculature to sustain homeostasis. Disturbance of this balance causes pathogenic angiogenesis and, especially in tumours, several activators such as VEGF are highly expressed in the tumour microenvironment and strongly induce tumour angiogenesis, the so-called angiogenic switch. Recently, we demonstrated that κ opioid receptor agonists function as anti-angiogenic factors, which impede the angiogenic switch, in vascular development and tumour angiogenesis by inhibiting the expression of receptors for VEGF. In clinical medicine, angiogenesis inhibitors that target VEGF signalling such as bevacizumab are used as anti-cancer drugs. Although therapies that inhibit tumour angiogenesis have been highly successful for tumour therapy, most patients eventually develop resistance to this anti-angiogenic therapy. Thus, we must identify novel targets for anti-angiogenic agents to sustain inhibition of angiogenesis for tumour therapy. The regulation of responses to κ opioid receptor ligands could be useful for controlling vascular formation under physiological conditions and in cancers, and thus could offer therapeutic benefits beyond the relief of pain., Linked Articles: This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2., (© 2014 The British Pharmacological Society.)

Upon removal of culture conditions that maintain an undifferentiated state, mouse embryonic stem cells (ESCs) differentiate into various cell types. Differentiation can be facilitated by forced expression of certain transcription factors (TFs), each of which can generally specify a particular developmental lineage. We previously established 137 mouse ESC lines, each of which carried a doxycycline-controllable TF. Among them, Sox9 has unique capacity: its forced expression accelerates differentiation of mouse ESCs into cells of all three germ layers. With the additional use of specific culture conditions, overexpression of Sox9 facilitated the generation of endothelial cells, hepatocytes and neurons from ESCs. Furthermore, Sox9 action increases formation of p21 (WAF1/CIP1), which then binds to the SRR2 enhancer of pluripotency marker Sox2 and inhibits its expression. Knockdown of p21 abolishes inhibition of Sox2 and Sox9-accelerated differentiation, and reduction of Sox2 2 days after the beginning of ESC differentiation can comparably accelerate mouse ESC formation of cells of three germ layers. These data implicate the involvement of the p21-Sox2 pathway in the mechanism of accelerated ESC differentiation by Sox9 overexpression. The molecular cascade could be among the first steps to program ESC differentiation., (© 2014. Published by The Company of Biologists Ltd.)

The origin and developmental mechanisms underlying coronary vessels are not fully elucidated. Here we show that myocardium-derived angiopoietin-1 (Ang1) is essential for coronary vein formation in the developing heart. Cardiomyocyte-specific Ang1 deletion results in defective formation of the subepicardial coronary veins, but had no significant effect on the formation of intramyocardial coronary arteries. The endothelial cells (ECs) of the sinus venosus (SV) are heterogeneous population, composed of APJ-positive and APJ-negative ECs. Among these, the APJ-negative ECs migrate from the SV into the atrial and ventricular myocardium in Ang1-dependent manner. In addition, Ang1 may positively regulate venous differentiation of the subepicardial APJ-negative ECs in the heart. Consistently, in vitro experiments show that Ang1 indeed promotes venous differentiation of the immature ECs. Collectively, our results indicate that myocardial Ang1 positively regulates coronary vein formation presumably by promoting the proliferation, migration and differentiation of immature ECs derived from the SV.

A network of transcription factors (TFs) determines cell identity, but identity can be altered by overexpressing a combination of TFs. However, choosing and verifying combinations of TFs for specific cell differentiation have been daunting due to the large number of possible combinations of ∼2,000 TFs. Here, we report the identification of individual TFs for lineage-specific cell differentiation based on the correlation matrix of global gene expression profiles. The overexpression of identified TFs-Myod1, Mef2c, Esx1, Foxa1, Hnf4a, Gata2, Gata3, Myc, Elf5, Irf2, Elf1, Sfpi1, Ets1, Smad7, Nr2f1, Sox11, Dmrt1, Sox9, Foxg1, Sox2, or Ascl1-can direct efficient, specific, and rapid differentiation into myocytes, hepatocytes, blood cells, and neurons. Furthermore, transfection of synthetic mRNAs of TFs generates their appropriate target cells. These results demonstrate both the utility of this approach to identify potent TFs for cell differentiation, and the unanticipated capacity of single TFs directly guides differentiation to specific lineage fates.

Opioids are effective analgesics for the management of moderate to severe cancer pain. Here we show that κ opioid receptor (KOR) agonists act as anti-angiogenic factors in tumors. Treatment with KOR agonists, U50,488H and TRK820, significantly inhibited human umbilical vein endothelial cell (HUVEC) migration and tube formation by suppressing VEGFR2 expression. In contrast, treatment with a μ opioid receptor agonist, DAMGO, or a δ opioid receptor agonist, SNC80, did not prevent angiogenesis in HUVECs. Lewis lung carcinoma (LLC) or B16 melanoma grafted in KOR knockout mice showed increased proliferation and remarkably enhanced tumor angiogenesis compared with those in wild type mice. On the other hand, repeated intraperitoneal injection of TRK820 (0.1-10 μg/kg, b.i.d.) significantly inhibited tumor growth by suppressing tumor angiogenesis. These findings indicate that KOR agonists play an important role in tumor angiogenesis and this knowledge could lead to a novel strategy for cancer therapy.

[This corrects the article DOI: 10.1371/journal.pone.0044368.].

Timing of cell differentiation is strictly controlled and is crucial for normal development and stem cell differentiation. However, underlying mechanisms regulating differentiation timing are fully unknown. Here, we show a molecular mechanism determining differentiation timing from mouse embryonic stem cells (ESCs). Activation of protein kinase A (PKA) modulates differentiation timing to accelerate the appearance of mesoderm and other germ layer cells, reciprocally correlated with the earlier disappearance of pluripotent markers after ESC differentiation. PKA activation increases protein expression of G9a, an H3K9 methyltransferase, along with earlier H3K9 dimethylation and DNA methylation in Oct3/4 and Nanog gene promoters. Deletion of G9a completely abolishes PKA-elicited acceleration of differentiation and epigenetic modification. Furthermore, G9a knockout mice show prolonged expressions of Oct3/4 and Nanog at embryonic day 7.5 and delayed development. In this study, we demonstrate molecular machinery that regulates timing of multilineage differentiation by linking signaling with epigenetics., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Although stem cell therapy is a promising strategy for cardiac restoration, the heterogeneity of transplanted cells has been hampering the precise understanding of the cellular and molecular mechanisms. Previously, we established a cardiovascular cell differentiation system from mouse pluripotent stem cells, in which cardiomyocytes (CMs), endothelial cells (ECs), and mural cells (MCs) can be systematically induced and purified. Combining this with cell sheet technology, we generated cardiac tissue sheets reassembled with defined cardiovascular populations. Here, we show the potentials and mechanisms of cardiac tissue sheet transplantation in cardiac function after myocardial infarction (MI). Transplantation of the cardiac tissue sheet to a rat MI model showed significant and sustained improvement of systolic function accompanied by neovascularization. Reduction of the infarct wall thinning and fibrotic length indicated the attenuation of left ventricular remodeling. Cell tracing with species-specific fluorescent in situ hybridization after transplantation revealed a relatively early loss of transplanted cells and an increase in endogenous neovascularization in the proximity of the graft, suggesting an indirect angiogenic effect of cardiac tissue sheets rather than direct CM contributions. We prospectively dissected the functional mechanisms with cell type-controlled sheet analyses. Sheet CMs were the main source of vascular endothelial growth factor. Transplantation of sheets lacking CMs resulted in the disappearance of neovascularization and subsequent functional improvement, indicating that the beneficial effects of the sheet were achieved by sheet CMs. ECs and MCs enhanced the sheet functions and structural integration. Supplying CMs to ischemic regions with cellular interaction could be a strategic key in future cardiac cell therapy., (Copyright © 2012 AlphaMed Press.)

Ets family protein Etv2 (also called ER71 or Etsrp) is a key factor for initiation of vascular and blood development from mesodermal cells. However, regulatory mechanisms and inducing signals for Etv2 expression have been largely unknown. Previously, we revealed that cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling enhanced differentiation of vascular progenitors into endothelial cells (ECs) and hematopoietic cells (HPCs) using an embryonic stem cell (ESC) differentiation system. Here, we show that PKA activation in an earlier differentiation stage can trigger EC/HPC differentiation through Etv2 induction. We found Etv2 was markedly upregulated by PKA activation preceding EC and HPC differentiation. We identified two cAMP response element (CRE) sequences in the Etv2 promoter and 5'-untranslated region and confirmed that CRE-binding protein (CREB) directly binds to the CRE sites and activates Etv2 transcription. Expression of a dominant negative form of CREB completely inhibited PKA-elicited Etv2 expression and induction of EC/HPCs from ESCs. Furthermore, blockade of PKA significantly inhibited Etv2 expression in ex vivo whole-embryo culture using Etv2-Venus knockin mice. These data indicated that PKA/CREB pathway is a critical regulator for the initiation of EC/HPC differentiation via Etv2 transcription. This early-stage molecular linkage between a triggering signal and transcriptional cascades for differentiation would provide novel insights in vascular and blood development and cell fate determination., (Copyright © 2012 AlphaMed Press.)

There is increasing evidence that functional crosstalk between GPCRs and EGFR contributes to the progression of colon, lung, breast, ovarian, prostate and head and neck tumors. In this study, we performed multiple analyses of GPCR expression in a gefitinib-resistant non-small cell lung cancer (NSCLC) cell line, H1975, which harbors an L858R/T790M mutation. To determine the expression profile of mRNAs encoding 384 GPCRs in normal human lung fibroblast (NHLF) and H1975 cells, a GPCR-specific microarray analysis was performed. A heat-map of the microarray revealed considerable differences in the expression of GPCRs between NHLF and H1975 cells. From the GPCR expression list, we selected some GPCR agonists/antagonist to investigate whether the respective ligands could affect the growth of H1975 cells. Among them, treatment with either a selective antagonist of adenosine A2a receptors, which were highly expressed in H1975 cell and another gefitinib-resistant NSCLC cells, HCC827GR cells or "small interfering RNA" (siRNA) targeting adenosine A2a receptors produced a significant decrease in cell viability of both H1975 and HCC827GR cells. Among up-regulated GPCRs in H1975 cells, Gs-, Gi- and Gq-coupled GPCRs were expressed almost equally. Among down-regulated GPCRs, Gi-coupled GPCRs were dominantly expressed in H1975 cells. The present results suggest that multilayered crosstalk between GPCRs and EGFR may play an important role in orchestrating downstream signaling molecules that are implicated in the development of gefitinib-resistant NSCLC.

The opioid system (opioid peptides and receptors) regulates a variety of neurophysiologic functions, including pain control. Here we show novel roles of the κ opioid system in vascular development. Previously, we revealed that cAMP/protein kinase A (PKA) signaling enhanced differentiation of vascular progenitors expressing VEGF receptor-2 (fetal liver kinase 1; Flk1) into endothelial cells (ECs) through dual up-regulation of Flk1 and Neuropilin1 (NRP1), which form a selective and sensitive VEGF(164) receptor. Kappa opioid receptor (KOR), an inhibitory G protein-coupled receptor, was highly expressed in embryonic stem cell-derived Flk1(+) vascular progenitors. The addition of KOR agonists to Flk1(+) vascular progenitors inhibited EC differentiation and 3-dimensional vascular formation. Activation of KOR decreased expression of Flk1 and NRP1 in vascular progenitors. The inhibitory effects of KOR were reversed by 8-bromoadenosine-3',5'-cAMP or a PKA agonist, N(6)-benzoyl-cAMP, indicating that KOR inhibits cAMP/PKA signaling. Furthermore, KOR-null or dynorphin (an endogenous KOR agonist)-null mice showed a significant increase in overall vascular formation and ectopic vascular invasion into somites at embryonic day -10.5. ECs in these null mice showed significant increase in Flk1 and NRP1, along with reciprocal decrease in plexinD1, which regulates vascular pathfinding. The opioid system is, thus, a new regulator of vascular development that simultaneously modifies 2 distinct vascular properties, EC differentiation and vascular pathfinding.

To investigate the effect of hypoxia or hypoxia/reoxygenation on vascular smooth muscle function, mechanical response of monkey coronary artery without endothelium was studied under normoxia, hypoxia, and hypoxia/reoxygenation. Hypoxia or hypoxia/reoxygenation impaired the relaxation by nitroglycerin or isosorbide dinitrate but not that by 8-bromoguanosine-3',5'-cyclic monophosphate or isoproterenol. Tempol restored the impaired relaxation by nitroglycerin or isosorbide dinitrate, but superoxide dismutase had no effect. Apocynin, an NADPH oxidase inhibitor, improved the nitroglycerin-induced relaxation under hypoxia, but not under reoxygenation. Under combined treatment of apocynin with oxypurinol (xanthine oxidase inhibitor), rotenone (mitochondria electron transport inhibitor), or both, hypoxic impairment of vasorelaxation was restored more effectively. Similarly, impairment of the nitroglycerin-induced vasorelaxation under hypoxia/reoxygenation was restored by combined treatment with three inhibitors, apocynin, oxypurinol, and rotenone. Increase in superoxide production under hypoxia tended to be inhibited by apocynin and that under hypoxia/reoxygenation was abolished by combined treatment with three inhibitors. These findings suggest that increased intracellular superoxide production under hypoxia or hypoxia/reoxygenation attenuates vasodilation mediated with a nitric oxide/soluble guanylyl cyclase, but not adenylyl cyclase, signaling pathway. The main source of superoxide production under hypoxia seems to be different from that under reoxygenation: superoxide is produced by NADPH oxidase during hypoxia, whereas it is produced by xanthine oxidase, mitochondria, or both during reoxygenation.[Supplementary Figure: available only at http://dx.doi.org/10.1254/jphs.11031FP].

Cyclic adenosine monophosphate (cAMP) is an important second messenger mediating physiological functions, including metabolism, gene expression, cell growth and differentiation. Recently, we demonstrated novel roles of cAMP pathway in endothelial cell (EC) differentiation and arterial-venous specification using an embryonic stem cell differentiation system. These studies offered a concept that vascular formation is accomplished by a 2-layered mechanism: (1) a basal mechanism for common EC differentiation, whereby vascular endothelial growth factor (VEGF) signaling plays a central role in the basal mechanism, and (2) a vascular diversification mechanism working on the basis of common EC differentiation. Vascular diversification, such as artery and vein formation, can be only achieved by enacting specific machineries in the presence of the basal EC machinery. cAMP/protein kinase A signaling contributes to common EC differentiation through upregulation of the VEGF-A receptors, Flk1 and neuropilin1. On the other hand, cAMP can activate phosphatidylinositol-3 kinase, which induces an arterial fate in vascular progenitors via dual activation of Notch and β-catenin signaling as an arterial-specific machinery. cAMP signaling thus plays a pivotal role in both the basal and diversification machinery during vascular development.

Molecular mechanisms controlling arterial-venous specification have not been fully elucidated. Previously, we established an embryonic stem cell differentiation system and demonstrated that activation of cAMP signaling together with VEGF induces arterial endothelial cells (ECs) from Flk1(+) vascular progenitor cells. Here, we show novel arterial specification machinery regulated by Notch and beta-catenin signaling. Notch and GSK3beta-mediated beta-catenin signaling were activated downstream of cAMP through phosphatidylinositol-3 kinase. Forced activation of Notch and beta-catenin with VEGF completely reconstituted cAMP-elicited arterial EC induction, and synergistically enhanced target gene promoter activity in vitro and arterial gene expression during in vivo angiogenesis. A protein complex with RBP-J, the intracellular domain of Notch, and beta-catenin was formed on RBP-J binding sites of arterial genes in arterial, but not venous ECs. This molecular machinery for arterial specification leads to an integrated and more comprehensive understanding of vascular signaling.

Fine tuning of vascular endothelial growth factor (VEGF) signaling is critical in endothelial cell (EC) differentiation and vascular development. Nevertheless, the system for regulating the sensitivity of VEGF signaling has remained unclear. Previously, we established an embryonic stem cell culture reproducing early vascular development using Flk1 (VEGF receptor-2)+ cells as common progenitors, and demonstrated that cyclic adenosine monophosphate (cAMP) enhanced VEGF-induced EC differentiation. Here we show that protein kinase A (PKA) regulates sensitivity of Flk1+ vascular progenitors to VEGF signaling for efficient EC differentiation. Blockade of PKA perturbed EC differentiation and vascular formation in vitro and ex vivo. Overexpression of constitutive active form of PKA (CA-PKA) potently induced EC differentiation and vascular formation. Expression of Flk1 and Neuropilin-1 (NRP1), which form a selective and sensitive receptor for VEGF(165), was increased only in CA-PKA-expressing progenitors, enhancing the sensitivity of the progenitors to VEGF(165) by more than 10 times. PKA activation induced the formation of a VEGF(165), Flk1, and NRP1 protein complex in vascular progenitors. These data indicate that PKA regulates differentiation potential of vascular progenitors to be endothelial competent via the dual induction of Flk1 and NRP1. This new-mode mechanism regulating "progenitor sensitivity" would provide a novel understanding in vascular development and regeneration.

Though cardiac progenitor cells should be a suitable material for cardiac regeneration, efficient ways to induce cardiac progenitors from embryonic stem (ES) cells have not been established. Extending our systematic cardiovascular differentiation method of ES cells, here we show efficient and specific expansion of cardiomyocytes and highly cardiogenic progenitors from ES cells. An immunosuppressant, cyclosporin-A (CSA), showed a novel effect specifically acting on mesoderm cells to drastically increase cardiac progenitors as well as cardiomyocytes by 10-20 times. Approximately 200 cardiomyocytes could be induced from one mouse ES cell using this method. Expanded progenitors successfully integrated into scar tissue of infracted heart as cardiomyocytes after cell transplantation to rat myocardial infarction model. CSA elicited specific induction of cardiac lineage from mesoderm in a novel mesoderm-specific, NFAT independent fashion. This simple but efficient differentiation technology would be extended to induce pluripotent stem (iPS) cells and broadly contribute to cardiac regeneration.

To compare the mechanisms underlying mechanical responses to ephedrine and Ephedra herb, a main component of Kakkon-to, in isolated male and female rabbit urinary bladder and urethral strips, responses of isolated strips to the agents were recorded in organ bath systems. Ephedrine and Ephedra herb relaxed the female urinary bladder to the similar extent. These relaxations are reversed to contractions by timolol. In the presence of timolol, ephedrine produced less contraction of urethral strips in the female than those in the male; this contraction was abolished by prazosin. Ephedra herb contracted the female urethra less than that of the male, and the contraction was stronger than that by ephedrine. The contraction caused by Ephedra herb in strips treated with timolol was significantly inhibited by prazosin. The prazosin-resistant contraction of the female urethra was greater than that of the male. Quinacrine, a phospholipase A(2) inhibitor, indomethacin, and AA861, a 5-lipoxygenase inhibitor, inhibited the contraction. The contraction was inhibited by ZK 158252, a leukotriene (LT) B(4)-receptor antagonist. These findings suggest that Ephedra herb contracts the urethra via arachidonic acid metabolites together with alpha(1)-adrenoceptor stimulation. The metabolites produced by 5-lipoxygenase may stimulate LTB(4), but not CysLt(1), receptors. These contractile components induced by Ephedra herb and Kakkon-to might be effective for the treatment of stress urinary incontinence.

We examined the mechanism of endothelial dysfunction in chronic renal failure (CRF), with reference to NO synthase. CRF was induced by 5/6 nephrectomy in rats. Either L-arginine (1.25 g/L in drinking water), tetrahydrobiopterin (BH4, 10 mg/kg per day in food), or a combination of the 2 were orally administered to CRF rats for 9 weeks. CRF rats showed elevation of systolic blood pressure compared with sham-operated rats. Endothelium-dependent relaxation induced by acetylcholine or A23187 in the isolated aorta was significantly reduced, and in vitro treatment with L-arginine, BH4, or superoxide dismutase restored the relaxation. Aortic segments from CRF rats showed significantly higher superoxide production in response to A23187, which was inhibited by L-NAME. Plasma concentrations of asymmetric dimethylarginine and symmetric dimethylarginine were higher in CRF rats. These changes in CRF rats were totally or partially decreased by L-arginine or BH4 supplementation in vivo. Interestingly, the combined treatment showed additive effects in certain parameters. These results suggest that vascular disorders in CRF rats may be partly due to NOS uncoupling caused by a relative deficiency of BH4 and partially due to accumulation of endogenous inhibitors of NOS and L-arginine uptake, resulting in the decrease of NO production and the increase of reactive oxygen species.