The precise and conceptual insight of circulating endothelial progenitor cell (EPC) kinetics is hampered by the absence of an assay system capable of evaluating the EPC differentiation cascade. An assay system for EPC colony formation was developed to delineate circulating EPC differentiation. EPC colony-forming assay using semisolid medium and single or bulk CD133؉ cells from umbilical cord blood exhibited the formation of two types of attaching cell colonies made of small or large cells featuring endothelial lineage potential and properties, termed small EPC colony-forming units and large EPC colony-forming units, respectively. In vitro and in vivo assays of each EPC colony-forming unit cell revealed a differentiation hierarchy from small EPC to large EPC colonies, indicating a primitive EPC stage with highly proliferative activity and a definitive EPC stage with vasculogenic properties, respectively. Experimental comparison with a conventional EPC culture assay system disclosed EPC colony-forming unit cells differentiate into noncolony-forming early EPC. The fate analysis of single CD133؉ cells into the endothelial and hematopoietic lineage was achieved by combining this assay system with a hematopoietic progenitor assay and demonstrated the development of colony-forming EPC and hematopoietic progenitor cells from a single hematopoietic stem cell. EPC colony-forming assay permits the determination of circulating EPC kinetics from single or bulk cells, based on the evaluation of hierarchical EPC colony formation. This assay further enables a proper exploration of possible links between the origin of EPC and hematopoietic stem cells, representing a novel and powerful tool to investigate the molecular signaling pathways involved in EPC biology. (Circ Res. 2011;109:20-37.) Key Words: clonogenic assay Ⅲ differentiation Ⅲ endothelial progenitor cell Ⅲ vasculogenesis D espite significant efforts in research and development with respect to endothelial progenitor cell (EPC) biology during the past 10 years after their initial isolation, 1 EPC remain a controversial topic among researchers because there is no definitive delineation of EPC, no clear differentiation hierarchy, or any unambiguously defined isolation protocol.EPC have been quantified and qualified either as cell populations identified by cell surface markers such as CD34, CD133, vascular endothelial growth factor receptor-2 (VEGFR-2), [1][2][3][4][5][6][7][8] or as adhesive cells 6,9,10 and colonies 11 using conventional EPC culture methods to produce spindle-shape adherent cells from peripheral blood (PB), bone marrow (BM), or umbilical cord blood (UCB) mononuclear cells (MNC) with endothelial growth factors and cytokines. These assays using conventional EPC culture protocols were simple and satisfactory to speculate on the vasculogenic properties of EPC-enriched fractions but have recently been criticized. These assays further group heterogeneous EPC into one qualitative category: "adhesive cultured EPC" without any hierarchical discrimination ...

The increased pulmonary vascular remodelling in Alk1(+/-) mice leads to signs of PH and is associated with eNOS-dependent ROS production, which is preventable by anti-oxidant treatment.

Closure of the ductus arteriosus requires prenatal formation of intimal cushions, which occlude the vessel lumen at birth. Survival of newborns with severe congenital heart defects, however, depends on ductal patency. We used a gene transfer approach to create a patent ductus arteriosus by targeting the fibronectin-dependent smooth muscle cell migration required for intimal cushion formation. Fetal lamb ductus arteriosus was transfected in utero with hemagglutinating virus of Japan liposomes containing plasmid encoding 'decoy' RNA to sequester the fibronectin mRNA binding protein. Fibronectin translation was inhibited and intimal cushion formation was prevented. We thus established the essential role of fibronectin-dependent smooth muscle cell migration in intimal cushion formation in the intact animal and the feasibility of incorporating biological engineering in the management of congenital heart disease.

Objective-Loss-of-function mutations in genes coding for transforming growth factor-␤/bone morphogenetic protein receptors and changes in nitric oxide) bioavailability are associated with hereditary hemorrhagic telangiectasia and some forms of pulmonary arterial hypertension. How these abnormalities lead to seemingly disparate pulmonary pathologies remains unknown. Endoglin (Eng), a transforming growth factor-␤ coreceptor, is mutated in hereditary hemorrhagic telangiectasia and involved in regulating endothelial NO• synthase (eNOS)-derived NO • production and oxidative stress. Because some patients with pulmonary arterial hypertension harbor ENG mutations leading to haplo insufficiency, we investigated the pulmonary vasculature of Eng ϩ/Ϫ mice and the potential contribution of abnormal eNOS activation to pulmonary arterial hypertension. Methods and Results-Hemodynamic, histological, and biochemical assessments and x-ray micro-CT imaging of adult Eng ϩ/Ϫ mice indicated signs of pulmonary arterial hypertension including increased right ventricular systolic pressure, degeneration of the distal pulmonary vasculature, and muscularization of small arteries. These findings were absent in 3-week-old Eng ϩ/Ϫ mice and were attributable to constitutively uncoupled eNOS activity in the pulmonary circulation, as evidenced by reduced eNOS/heat shock protein 90 association and increased eNOS-derived superoxide () production in a BH 4 -independent manner. These changes render eNOS unresponsive to regulation by transforming growth factor-␤/bone morphogenetic protein and underlie the signs of pulmonary arterial hypertension that were prevented by Tempol. Conclusion-Adult Engϩ/Ϫ mice acquire signs of pulmonary arterial hypertension that are attributable to uncoupled eNOS activity and increasedϪ production, which can be prevented by antioxidant treatment. Eng links transforming growth factor/bone morphogenetic protein receptors to the eNOS activation complex, and its reduction in the pulmonary vasculature leads to increased oxidative stress and pulmonary arterial hypertension. Key Words: Alk-1 Ⅲ endoglin Ⅲ free radicals Ⅲ nitric oxide Ⅲ pulmonary arterial hypertension Ⅲ transforming growth factor E ndoglin (Eng; CD105) is a an ancillary receptor for several transforming growth factor (TGF)-␤ superfamily ligands, including bone morphogenetic proteins (BMP). 1 It is predominantly expressed on vascular endothelial cells 2 and found in both TGF-␤ and BMP receptor complexes, 1,3 where it modulates TGF-␤1/␤3 4 and BMP9/10 effects, 5 respectively, via its physical association with the activin-like kinase receptor-1 (ACVLR1) gene product, ALK1. Eng-null mice die at mid-gestation with impaired angiogenesis and severe cardiac defects. 6,7 Whereas Eng ϩ/Ϫ mice have a normal lifespan, they display abnormal systemic vascular autoregulatory functions related to endothelial nitric oxide synthase (eNOS) activity. 8 Mutations in the endoglin (ENG) and activin-like kinase 1 receptor (ACVLR1) genes lead to haploinsufficiency and are the underlying ca...

Key Points Notch signaling controls hematopoiesis of human pluripotent stem cells.

Inherited vascular malformations are commonly autosomal dominantly inherited with high, but incomplete, penetrance; they often present as multiple lesions. We hypothesized that Knudson's two-hit model could explain this multifocality and partial penetrance. We performed a systematic analysis of inherited glomuvenous malformations (GVMs) by using multiple approaches, including a sensitive allele-specific pairwise SNP-chip method. Overall, we identified 16 somatic mutations, most of which were not intragenic but were cases of acquired uniparental isodisomy (aUPID) involving chromosome 1p. The breakpoint of each aUPID is located in an A- and T-rich, high-DNA-flexibility region (1p13.1-1p12). This region corresponds to a possible new fragile site. Occurrences of these mutations render the inherited glomulin variant in 1p22.1 homozygous in the affected tissues without loss of genetic material. This finding demonstrates that a double hit is needed to trigger formation of a GVM. It also suggests that somatic UPID, only detectable by sensitive pairwise analysis in heterogeneous tissues, might be a common phenomenon in human cells. Thus, aUPID might play a role in the pathogenesis of various nonmalignant disorders and might explain local impaired function and/or clinical variability. Furthermore, these data suggest that pairwise analysis of blood and tissue, even on heterogeneous tissue, can be used for localizing double-hit mutations in disease-causing genes.

Hemodynamic unloading of lungs with pulmonary vascular disease results in progressive normalization of pulmonary artery structure. These results are the first to provide a rationale for attempting to induce regression of pulmonary vascular disease by pressure unloading of the pulmonary circulation. Methods to mechanically unload the pulmonary circulation should be critically evaluated as a strategy for staged surgical repair of congenital heart defects despite presumed irreversible pulmonary hypertension.

, and clinical signs of disease are generally more evident later in life. We previously showed that systemic vessels of adult Eng heterozygous (Eng ϩ/Ϫ ) mice exhibit increased vasorelaxation due to uncoupling of endothelial nitric oxide synthase (eNOS). We postulated that these changes may develop with age and evaluated pulmonary arteries from newborn and adult Eng ϩ/Ϫ mice for eNOS-dependent, acetylcholine (ACh-induced) vasorelaxation, compared with that of age-matched littermate controls. While ACh-induced vasorelaxation was similar in all newborn mice, it was significantly increased in the adult Eng ϩ/Ϫ vs. control vessels. The vasodilatory responses were inhibited by L-NAME suggesting eNOS dependence. eNOS uncoupling was observed in lung tissues of adult, but not newborn, heterozygous mice and was associated with increased production of reactive O2 species (ROS) in adult Eng ϩ/Ϫ vs. control lungs. Interestingly, ROS generation was higher in adult than newborn mice and so were the levels of NADPH oxidase 4 and SOD 1, 2, 3 isoforms. However, enzyme protein levels and NADPH activity were normal in adult Eng ϩ/Ϫ lungs indicating that the developmental maturation of ROS generation and scavenging cannot account for the increased vasodilatation observed in adult Eng ϩ/Ϫ mice. Our data suggest that eNOS-dependent H2O2 generation in Eng ϩ/Ϫ lungs accounts for the heightened pulmonary vasorelaxation. To the extent that these mice mimic human HHT1, age-associated pulmonary vascular eNOS uncoupling may explain the late childhood and adult onset of clinical lung manifestations. lung; newborn; pulmonary vascular resistance; hereditary hemorrhagic telangiectasia LUNG BLOOD FLOW IS MOSTLY dependent on the regional arteriolar and venular intraluminal diameter that ultimately determines the pulmonary vascular resistance (PVR). Nitric oxide (NO) is constitutively produced by endothelial and smooth muscle cells (39) via synthases (NOS) of which there are three isoforms: endothelial (eNOS), neuronal (nNOS), and inducible (iNOS). Although there is evidence that iNOS and nNOS are expressed in the fetal pulmonary vasculature (38), lung eNOS protein expression increases during gestation suggesting that its vascular tissue content and/or activity is in part responsible for the high PVR prenatally and the changes occurring after birth (15). In sheep, lung eNOS expression is maximal in late gestation (34), whereas in rats it is highest either before (33), or immediately after, birth (23). Postnatally, lung vascular tissue eNOS expression was shown to decrease with age in pigs (17).eNOS converts L-arginine to L-citrulline to generate NO. Its activity is dependent on Ca 2ϩ /calmodulin (CaM), but also on subcellular localization, posttranslational modifications, and interaction with several regulatory proteins, including Hsp90 (11,14). Hsp90 facilitates CaM-induced release from caveolae and acts as a scaffold factor for eNOS; it is necessary for eNOS phosphorylation at Ser1177 (13, 43). This complex process controls the state of eNOS ac...