Losartan ameliorates "upstream" pulmonary vein vasculopathy in a piglet model of pulmonary vein stenosis.

OBJECTIVES: Pulmonary vein stenosis (PVS) is a relentless disease with a poor prognosis. Although surgical repair can effectively treat "downstream" (near left atrial junction) PVS, residual "upstream" (deep in lung parenchyma) PVS commonly dictates long-term survival. Our initial studies revealed an association between PVS and transforming growth factor-ß signaling, which led us to investigate the effect of losartan on upstream pulmonary vein vasculopathy in a piglet model of PVS. METHODS: Neonatal Yorkshire piglets underwent sham surgical banding (sham, n = 6), staged bilateral pulmonary vein banding of all pulmonary veins except the right middle pulmonary vein (banded, n = 6), and staged pulmonary vein banding with losartan treatment (losartan, 1 mg/kg/d, n = 7). After 7 weeks, the hemodynamic data were obtained and the piglets killed. RESULTS: Pulmonary vein banding (compared with sham) was associated with continuous turbulent flow in banded pulmonary veins, pulmonary hypertension (pulmonary artery/systemic blood pressure ratio 0.51 ± 0.06 vs 0.23 ± 0.02, P < .001), and diffuse pulmonary vein intimal hyperplasia in the upstream pulmonary veins (P < .001). Losartan administration decreased the pulmonary artery/systemic blood pressure ratios compared with those in the banded piglets (0.36 ± 0.08 vs 0.51 ± 0.06, P = .007) but it remained greater than those in the sham group (P = .001). Losartan was also associated with diminished pulmonary vein intimal hyperplasia compared with that in the banded piglets (P < .001) but still remained more than that in the sham group (P = .035). Pulmonary vein banding reduced vascular endothelial-cadherin expression, indicative of diminished endothelial integrity, which was restored with losartan. CONCLUSIONS: Losartan treatment improved PVS-associated pulmonary hypertension and intimal hyperplasia and might be a beneficial prophylactic therapy for patients at high risk of developing PVS after pulmonary vein surgery.

The neural stem cell lineage reveals novel relationships among spermatogonial germ stem cells and other pluripotent stem cells.

The embryonic stem cell (ESC) derived from the inner cell mass is viewed as the core pluripotent cell (PC) type from which all other cell types emanate. This familiar perspective derives from an embryological time line in which PCs are ordered according to their time of appearance. However, this schema does not take into account their potential for interconversion, thereby excluding this critical quality of PCs. The persistence of bona fide pluripotent adult stem cells has garnered increasing attention in recent years. Adult pluripotent spermatogonial germ stem cells (aSGSCs) arise from primordial germ cells (pGCs) that emerge from the epiblast during gastrulation. Adult definitive neural stem cells (dNSCs) arise clonally from pluripotent embryonic primitive neural stem cells (pNSCs), which can also be derived clonally from ESCs. To test for stem cell-type convertibility, we employed differentiation in the clonal lineage from ESCs to pNSCs to dNSCs, and revealed the relationships and lineage positioning among various PC populations, including spermatogonial germ cells (aSGSCs), epiblast-derived stem cells (Epi-SCs) and the bFGF, Activin, and BIO-derived stem cell (FAB-SC). Adult, murine aSGSCs assumed a 'pseudo-ESC' state in vitro, and then differentiated into dNSCs, but not pNSCs. Similarly, Epi-SCs and FAB-SCs only gave rise to dNSCs and not to pNSCs. The results of these experiments suggest a new pluripotency lineage model describing the relationship(s) among PCs that better reflects the transitions between these cell types in vitro.

Ontogeny of human umbilical cord perivascular cells: molecular and fate potential changes during gestation.

Human umbilical cord-derived perivascular cells (PVCs) are a recently characterized source of mesenchymal stromal cells that has gained much interest in the field of cellular therapeutics. However, very little is known about the changes in fate potential and restrictions that these cells undergo during gestational development. This study is the first to examine the phenotypic, molecular, and functional properties of first trimester (FTM)-derived PVCs, outlining properties that are unique to this population when compared to term (TERM) counterparts. FTM- and TERM-PVCs displayed analogous mesenchymal, perivascular, and immunological immunophenotypes. Both PVCs could be maintained in culture without alteration to these phenotypes or mesenchymal lineage differentiation potential. Some unique features of FTM-PVCs were uncovered in this study: (1) while the gene signatures of FTM- and TERM-PVCs were similar, key differences were observed, namely, that the Oct4A and Sox17 proteins were detected in FTM-PVCs, but not in TERM counterparts; (2) FTM-PVCs exhibited a greater proliferative potential; and (3) FTM-PVCs were more efficient in their in vitro differentiation toward selective mesenchymal cell types, including the chondrogenic and adipogenic lineages, as well as toward neuronal- and hepatocyte-like lineages, when compared to TERM-PVCs. Both PVCs were able to generate osteocytes and cardiomyocyte-like cells with similar efficiencies in vitro. Overall, FTM-PVCs show more plasticity than TERM-PVCs with regard to fate acquisition, suggesting that a restriction in multipotentiality is imposed on PVCs as gestation progresses. Taken together, our findings support the idea that PVCs from earlier in gestation may be better than later sources of multipotent stromal cells (MSCs) for some regenerative medicine applications.

Endothelial nitric oxide synthase gene expression during murine embryogenesis: commencement of expression in the embryo occurs with the establishment of a unidirectional circulatory system.

To elucidate the role of endothelial NO synthase (eNOS)-derived NO during mammalian embryogenesis, we assessed the expression of the eNOS gene during development. Using transgenic eNOS promoter/reporter mice (with beta-galactosidase and green fluorescent protein reporters), in situ cRNA hybridization, and immunohistochemistry to assess transcription, steady-state mRNA levels, and protein expression, respectively, we noted that eNOS expression in the developing cardiovascular system was highly restricted to endothelial cells of medium- and large-sized arteries and the endocardium. The onset of transcription of the native eNOS gene and reporters coincided with the establishment of robust, unidirectional blood flow at embryonic day 9.5, as assessed by Doppler ultrasound biomicroscopy. Interestingly, reporter transgene expression and native eNOS mRNA were also observed in discrete regions of the developing skeletal musculature and the apical ectodermal ridge of developing limbs, suggesting a role for eNOS-derived NO in limb development. In vitro studies of promoter/reporter constructs indicated that similar eNOS promoter regions operate in both embryonic skeletal muscle and vascular endothelial cells. In summary, transcriptional activity of the eNOS gene in the murine circulatory system occurred following the establishment of embryonic blood flow. Thus, the eNOS gene is a late-onset gene in endothelial ontogeny.

Relative reduction of endothelial nitric-oxide synthase expression and transcription in atherosclerosis-prone regions of the mouse aorta and in an in vitro model of disturbed flow.

Atherosclerosis develops in distinct regions of the arterial tree. Defining patterns and mechanisms of endothelial cell gene expression in different regions of normal arteries is key to understanding the initial molecular events in atherogenesis. In this study, we demonstrated that the expression of endothelial nitric-oxide synthase (eNOS), an atheroprotective gene, and its phosphorylation on Ser(1177), a marker of activity, were lower in regions of the normal mouse aorta that are predisposed to atherosclerosis. The same expression pattern was observed in mouse strains that are both susceptible and resistant to atherosclerosis, and the topography of eNOS expression was inverse to p65, the main nuclear factor-kappaB subunit. Modeling of disturbed and uniform laminar flow in vitro reproduced the expression patterns of eNOS and p65 that were found in vivo. Heterogeneous nuclear RNA expression and RNA polymerase II chromosome immunoprecipitation studies demonstrated that regulation of transcription contributed to increased eNOS expression in response to shear stress. In vivo, the transcription of eNOS was reduced in regions of the mouse aorta predisposed to atherosclerosis, as defined by reporter gene expression in eNOS promoter-beta-galactosidase reporter transgenic mice. These data suggest that disturbed hemodynamic patterns found at arterial branches and curvatures uniquely modulate endothelial cell gene expression by regulating transcription, potentially explaining why these regions preferentially develop atherosclerosis when risk factors such as hypercholesterolemia are introduced.

The cell-specific expression of endothelial nitric-oxide synthase: a role for DNA methylation.

The basis for the endothelial cell-restricted expression of endothelial nitric-oxide synthase (eNOS) is not known. While transgenic promoter/reporter mice demonstrated endothelium cell-specific eNOS expression, we found robust expression of episomal eNOS promoter/reporter constructs in cell types that do not express the native eNOS transcript. To explore the mechanism underlying this differential activity pattern of chromatin-versus episome-based eNOS promoters, we examined the methylation status of 5'-regulatory sequences of the human eNOS gene. DNA methylation differed dramatically between endothelial and nonendothelial cell types, including vascular smooth muscle cells. This same cell type-specific methylation pattern was observed in vivo in endothelial and vascular smooth muscle cells of the mouse aorta at the native murine eNOS promoter. We addressed the functional consequences of methylation on eNOS transcription using transient transfection of in vitro methylated promoter/reporter constructs and found that methylated constructs exhibited a marked decrease in the synergistic action of Sp1, Sp3, and Ets1 on eNOS promoter activity. The addition of methyl-CpG-binding protein 2 further reduced the transcriptional activity of methylated eNOS constructs. Importantly, chromatin immunoprecipitation demonstrated the presence of Sp1, Sp3, and Ets1 at the native eNOS promoter in endothelial cells but not in vascular smooth muscle cells. Finally, robust expression of eNOS mRNA was induced in nonendothelial cell types following inhibition of DNA methyltransferase activity with 5-azacytidine, demonstrating the importance of DNA methylation-mediated repression. This report is the first to show that promoter DNA methylation plays an important role in the cell-specific expression of a constitutively expressed gene in the vascular endothelium.

An alternative promoter of the human neuronal nitric oxide synthase gene is expressed specifically in Leydig cells.

Neuronal nitric oxide synthase (nNOS) plays a modulatory role in the biology of a variety of neuroendocrine tissues and is especially relevant to gonadal function. We have previously reported the cloning and characterization of a variant of the nNOS protein, termed testis nNOS (TnNOS), the mRNA for which was restricted in expression to male gonadal tissues. To examine the cell-specificity of the testis-specific NOS regulatory regions we defined patterns of beta-galactosidase expression of an insertional transgene in which the reporter gene lacZ was under the transcriptional control of the human TnNOS promoter. beta-galactosidase activity was detected exclusively in the interstitial cells of the testis in transgenic mice. These cells also evidenced positive staining for nNOS protein and were identified as androgen-producing Leydig cells by staining with the Leydig cell marker, P(450)scc. Expression of the promoter was absent in cells of the seminiferous tubules, specifically germline cells of different stages and Sertoli cells. In contrast to the male gonad, beta-galactosidase activity was not detected in ovaries of adult female mice. Activity was also not evident in organs known to express full-length nNOS, such as skeletal muscle, kidney, or cerebellum. The same pattern of beta-galactosidase staining was observed in independent transgenic founders and was distinct from that observed for an endothelial NOS promoter/reporter transgene. In the testis of male adult eNOS promoter-reporter transgenic mice, beta-galactosidase activity was expressed only in endothelial cells of large- and medium-sized arterial blood vessels. Transcriptional activity of the human TnNOS promoter could not be detected in a variety of cell types, including Leydig cells, using episomal promoter-reporter constructs suggesting that a nuclear environment and higher order genomic complexity are required for appropriate promoter function. The restricted expression pattern of an nNOS variant in Leydig cells of the male gonad suggests an important role in the regulation of testosterone release and represents an intriguing model with which to dissect the molecular basis of Leydig cell-specific gene expression.