Signaling via PI3K/FOXO1A pathway modulates formation and survival of human embryonic stem cell-derived endothelial cells

Vascular derivatives of human embryonic stem cells (hESC) are being developed as sources of tissue-specific cells for organ regeneration. However, identity of developmental pathways that modulate the specification of endothelial cells is not known yet. We studied phosphatidylinositol 3-kinase (PI3K)-Forkhead box O transcription factor 1A (FOXO1A) pathways during differentiation of hESC toward endothelial lineage and on proliferation, maturation, and cell death of hESC-derived endothelial cells (hESC-EC). During differentiation of hESC, expression of FOXO1A transcription factor was linked to the expression of a cluster of angiogenesis- and vascular remodeling-related genes. PI3K inhibitor LY294002 activated FOXO1A and induced formation of CD31(+) hESC-EC. In contrast, differentiating hESC with silenced FOXO1A by small interfering RNA (siRNA) showed lower mRNA levels of CD31 and angiopoietin2. LY294002 decreased proliferative activity of purified hESC-EC, while FOXO1A siRNA increased their proliferation. LY294002 inhibits migration and tube formation of hESC-EC; in contrast, FOXO1A siRNA increased in vitro tube formation activity of hESC-EC. After in vivo conditioning of cells in athymic nude rats, cells retain their low FOXO1A expression levels. PI3K/FOXO1A pathway is important for function and survival of hESC-EC and in the regulation of endothelial cell fate. Understanding these properties of hESC-EC may help in future applications for treatment of injured organs

High rate of in-stent restenosis after coronary intervention in carriers of the mutant mannose-binding lectin allele

BACKGROUND: In-stent restenosis occurs in 10-30% of patients following bare metal stent (BMS) implantation and has various risk factors. Mannose-binding lectin (MBL) is known to have effect on the progression of atherosclerosis. Single nucleotide polymorphisms (SNP) of the MBL2 gene intron 1 (codon 52, 54, 57) are known to modulate the bioavailability of the MBL protein. Our aim was to identify the association of these polymorphisms of the MBL gene in the occurrence of in-stent restenosis after coronary artery bare metal stent implantation. METHODS: In a non-randomized prospective study venous blood samples were collected after recoronarography from 225 patients with prior BMS implantation. Patients were assigned to diffuse restenosis group and control group based on the result of the coronarography. MBL genotypes were determined using quantitative real-time PCR. Proportion of different genotypes was compared and adjusted with traditional risk factors using multivariate logistic regression. RESULTS: Average follow-up time was 1.0 (+ - 1.4) year in the diffuse restenosis group (N = 117) and 2.7 (+ - 2.5) years in the control group (N = 108). The age, gender distribution and risk status was not different between study groups. Proportion of the MBL variant genotype was 26.8% (29 vs. 79 normal homozygous) in the control group and 39.3% (46 vs. 71 normal homozygous) in the restenosis group (p = 0.04). In multivariate analysis the mutant allele was an independent risk factor (OR = 1.96, p = 0.03) of in-stent restenosis. CONCLUSIONS: MBL polymorphisms are associated with higher incidence of development of coronary in-stent restenosis. The attenuated protein function in the mutant allelic genotype may represent the underlying mechanism

Pathogen Sensing Pathways in Human Embryonic Stem Cell Derived-Endothelial Cells: Role of NOD1 Receptors.

Human embryonic stem cell-derived endothelial cells (hESC-EC), as well as other stem cell derived endothelial cells, have a range of applications in cardiovascular research and disease treatment. Endothelial cells sense Gram-negative bacteria via the pattern recognition receptors (PRR) Toll-like receptor (TLR)-4 and nucleotide-binding oligomerisation domain-containing protein (NOD)-1. These pathways are important in terms of sensing infection, but TLR4 is also associated with vascular inflammation and atherosclerosis. Here, we have compared TLR4 and NOD1 responses in hESC-EC with those of endothelial cells derived from other stem cells and with human umbilical vein endothelial cells (HUVEC). HUVEC, endothelial cells derived from blood progenitors (blood outgrowth endothelial cells; BOEC), and from induced pluripotent stem cells all displayed both a TLR4 and NOD1 response. However, hESC-EC had no TLR4 function, but did have functional NOD1 receptors. In vivo conditioning in nude rats did not confer TLR4 expression in hESC-EC. Despite having no TLR4 function, hESC-EC sensed Gram-negative bacteria, a response that was found to be mediated by NOD1 and the associated RIP2 signalling pathways. Thus, hESC-EC are TLR4 deficient but respond to bacteria via NOD1. This data suggests that hESC-EC may be protected from unwanted TLR4-mediated vascular inflammation, thus offering a potential therapeutic advantage

Right Ventricular Adaptation Is Associated with the Glu298Asp Variant of the NOS3 Gene in Elite Athletes

Nitric oxide (NO), an important endogenous pulmonary vasodilator is synthetized by the endothelial NO synthase (NOS3). Reduced NO bioavailability and thus the Glu298Asp polymorphism of NOS3 may enhance right ventricular (RV) afterload and hypertrophic remodeling and influence athletic performance. To test this hypothesis world class level athletes (water polo players, kayakers, canoeists, rowers, swimmers, n = 126) with a VO2 maximum greater than 50ml/kg/min were compared with non-athletic volunteers (n = 155). Cardiopulmonary exercise tests and cardiac magnetic resonance imaging (cMRI) were performed to determine structural or functional changes. Genotype distribution of the NOS3 Glu298Asp polymorphism was not affected by gender or physical performance. Cardiac MRI showed increased stroke volume with eccentric hypertrophy in all athletes regardless of their genotype. However, the Asp allelic variant carriers had increased RV mass index (32+/-6g versus 27+/-6g, p<0.01) and larger RV stroke volume index (71+/-10ml versus 64+/-10ml, p<0.01) than athletes with a Glu/Glu genotype. Genotype was not significantly associated with athletic performance. In the non-athletic group no genotype related differences were detected. The association between the NOS3 Glu298Asp polymorphism and RV structure and dimension in elite athletes emphasizes the importance of NOS3 gene function and NO bioavailability in sport related cardiac adaptation

Simultaneous effect of habitat and age on reproductive success of imperial eagle (Aquila heliaca) in Hungary.

Abstract An unexpected expansion of the Eastern Imperial Eagle (Aquila heliaca) population was observed in East Hungary from mountainous habitat into lowlands from 1989 onwards. Here the population markedly increased from 2 to 59 breeding pairs by 2006, while the mountainous population remained more or less stable with 12-17 breeding pairs. At the beginning of the expansion process the nearest neighbour distances between breeding pairs was lower in the mountains than in the lowlands, but presently they are similar, indicating a saturation process in the lowland areas, but no density dependence was revealed on breeding success. During the study period a higher ratio of non-adult pairs was observed in the lowland territories (49%) than in the mountains (22%). We found that both age and habitat influenced breeding success. We also found that age-effect was significant on success rate (i.e. the ratio of pairs that produce at least one chick), while habitat-effect was more evident on fledging success (i.e. the number of fledglings per productive pair). The overall productivity (i.e. number of fledglings per breeding pair) was affected primarily by the age of the pairs, but the interaction term of age x habitat also was significant. We suppose that better feeding possibilities (closer foraging areas and larger prey density) could explain the higher fledging success in the lowlands. We also predicted that pairs inhabiting agricultural areas in the lowlands will have a reduced success rate due to higher human disturbance, together with an age effect of the breeding pairs. Therefore adult pairs probably can habituate to disturbance even if it happens in the close vicinity of their nesting sites.</jats:p

Alterations of the perivascular dystrophin-dystroglycan complex following brain lesions. An immunohistochemical study in rats

Dystroglycan is a laminin receptor, which with dystrophins and other components forms the dystrophin-dystroglycan complex. It has an important role in the formation of gliovascular connections, cerebral vascularisation and blood-brain barrier. Dystroglycan consists of two sub-units, α and ß. Previous studies demonstrated that the ß-dystroglycan immunoreactivity of cerebral vessels temporarily disappeared in the area adjacent to the lesion, whereas the vascular laminin which is not immunoreactive in the intact brain became detectable. The present study extends these investigations over other components of the complex: utrophin, α1-syntrophin and α1-dystrobrevin. The experiments were performed on adult rats. The lesions were stab wounds or cryogenic lesions in deep ketamine-xylasine narcosis. Following survival periods 2 to 30 days, the animals were perfused and floating brain sections were processed for fluorescent immunohistochemistry. The α1-dystrobrevin, like ß-dystroglycan, vanished temporarily around the lesion. The immunoreactivity of utrophin changed in a similar way to that of laminin. In intact brains they were confined to the entering segments of the vessels and to the circumventricular organs. Following lesions their immunoreactivity manifested in the vessels around the lesions. However, utrophin followed laminin with a delay: their peaks were about POD (postoperative days) 21 and 7, respectively. Only immunoreactivity of α1-syntrophin appeared in the reactive astrocytes, peaking at POD 14. Double-labeling proved its co-localization with GFAP. Cryogenic lesions had similar immunohistochemical effects, but provided more suitable samples for Western blot analysis, which proved the altered levels of α1-dystrobrevin and α1-syntrophin. The phenomena may help to monitor the post-lesion vascular processes and the alterations of the gliovascular connections

Components of the Basal Lamina and Dystrophin–Dystroglycan Complex in the Neurointermediate Lobe of Rat Pituitary Gland: Different Localizations of β-Dystroglycan, Dystrobrevins, α1-Syntrophin, and Aquaporin-4

The so-called neurointermediate lobe is composed of the intermediate and neural lobes of the pituitary. The present immunohistochemical study investigated components of the basal lamina (laminin, agrin, and perlecan), the dystrophin–dystroglycan complex (dystrophin, β-dystroglycan, α1-dystrobrevin, β-dystrobrevin, utrophin, and α1-syntrophin), and the aquaporins (aquaporin-4 and -9). Glia markers (GFAP, S100, and glutamine synthetase) and components of connective tissue (collagen type I and fibronectin) were also labeled. In the neurohypophysis, immunostaining of basal lamina delineated meningeal invaginations. In these invaginations, vessels were seen to penetrate the organ without submerging into its parenchyma. On the parenchymal side of the invaginations, β-dystroglycan was detected, whereas utrophin was detected in the walls of vessels. Immunostaining of α1-dystrobrevin and α1-syntrophin did not delineate the vessels. The cells of the intermediate lobe were fully immunoreactive to α1-dystrobrevin and α1-syntrophin, whereas components of the basal lamina delineated the contours of the cells. GFAP-immunoreactive processes surrounded them. Aquaporin-4 localized at the periphery of the neurohypophysis, mainly adjacent to the intermediate lobe but not along the vessels. It colocalized only partially with GFAP and not at all with α1-syntrophin. Aquaporin-9 was not detected. These results emphasize the possibility that the components of the dystrophin–dystroglycan complex localize differently and raise the question about the roles of dystrobrevins, α1-syntrophin, and aquaporin-4 in the functions of the intermediate and neural lobes, respectively. (J Histochem Cytochem 58:463–479, 2010