HDL is essential for atherosclerotic lesion regression in Apoe knockout mice by bone marrow Apoe reconstitution.

BACKGROUND AND AIMS: Although studies in mice have suggested that lesion regression is feasible, the underlying mechanisms remain largely unknown. Here we determined the impact of high-density lipoprotein (HDL) on atherosclerosis regression outcome. METHODS: Atherosclerotic lesion dynamics were studied upon bone marrow transplantation-mediated re-introduction of apolipoprotein E (Apoe) in Apoe knockout mice. Probucol was used to pharmacologically deplete HDL. RESULTS: Restoration of Apoe function was associated with an initial growth of atherosclerotic lesions and parallel decrease in lesional macrophage foam cell content (47 ±â€¯4% at 4 weeks versus 72 ±â€¯2% at baseline: p < 0.001), despite the fact that cholesterol levels were markedly reduced. Notably, significant lesion regression was detected from 4 weeks onwards, when plasma cholesterol levels had returned to the normolipidemic range. As a result, lesions were 41% smaller (p < 0.05) at 8 weeks than at 4 weeks after bone marrow transplantation. Regressed lesions contained an even lower level of macrophage foam cells (33 ±â€¯5%: p < 0.001) and were rich in collagen. Probucol co-treatment was associated with a 3.2-fold lower (p < 0.05) plasma HDL-cholesterol level and a more pro-inflammatory (CCR2+) monocyte phenotype. Importantly, probucol-treated mice exhibited atherosclerotic lesions that were larger than those of regular chow diet-fed bone marrow transplanted mice at 8 weeks (186 ± 15*103 µm2 for probucol-treated versus 120 ± 19*103 µm2 for controls: p < 0.05). CONCLUSIONS: We have shown that probucol-induced HDL deficiency impairs the ability of established lesions to regress in response to reversal of the genetic hypercholesterolemia in Apoe knockout mice. Our studies thus highlight a crucial role for HDL in the process of atherosclerosis regression.

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.

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.

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.

High-volume lesions using a new second-generation open irrigation radiofrequency catheter are associated with the development of inhomogeneous lesions.

BACKGROUND: After catheter ablation there is often a discrepancy between acute and chronic success rates. We aimed to evaluate major determinants for lesion quality and understand different manifestations of lesion structures. METHODS: In a canine thigh muscle model radiofrequency (RF) current was delivered for 60 seconds at 30 W (n = 39) or 50 W (n = 18) with 15-g contact force. A second-generation 12-hole gold open irrigation catheter (SGIT) and a first-generation six-hole platinum-iridium catheter (FGIT; Biotronik, Berlin, Germany) were used. Electrode and tissue temperatures (at the surface and 3.5-mm and 7-mm depth) were recorded and lesion dimensions were measured. Lesions with steam pops were excluded. Histological examination was performed to evaluate homogeneity of the lesions. Inhomogeneity was defined as a visual multiband lesion pattern indicating different histological characteristics. RESULTS: In total 57 lesions were created. Seventeen lesions were excluded (steam pops) and 40 lesions were analyzed. A total number of 11 homogeneous and 29 inhomogeneous lesions were identified. Using the SGIT catheter 16.7% of the lesions was homogeneous and 83.3% inhomogeneous; for FGIT it was 43.8% and 56.2% (P = 0.065), respectively. Homogeneous lesions had lower volumes as compared to inhomogeneous lesions (514.0 ± 198.8 vs 914.8 ± 399.1 mm, P = 0.003). Multiple logistic regression analysis indicated that the SGIT catheter is a significant predictor for inhomogeneous lesions (odds ratio 6.5, 95% confidence interval 1.1-38.8; P = 0.040) independent from power setting and flow rate. CONCLUSIONS: The development of inhomogeneous lesions after acute RF ablation is associated with higher lesion volumes and the use of the second-generation irrigation gold-tip catheter.

Immunosuppressive agents modulate function, growth, and survival of cardiomyocytes and endothelial cells derived from human embryonic stem cells.

Cardiac cell replacement therapy by using human embryonic stem cell (hESC) derivatives remains a potential approach to regenerate myocardium. The major hurdles to clinical application of this technology are immunogenicity and post-transplantation cell death. Here we examined the effects of calcineurin-targeting immunosuppressants cyclosporine A (CsA) and FK506, as well as rapamycin and a selective inhibitor of calcineurin-binding downstream nuclear factor of activated T-cell (NFAT) transcription factor VIVIT on the proliferative activity, function, and survival of hESC-derived cardiomyocytes (hESC-CM) and endothelial cells (hESC-EC) in culture. As shown by automated microscopy, treatments with CsA, FK506, and rapamycin all decreased proliferation, reducing the percentage of hESC-CM and hESC-EC with the mitotic marker Ki67(+) by as much as 60% and 74%, respectively. Administration of the cell permeable analogue 11R-VIVIT protein did not modulate their proliferative activity. All immunosuppressants reversed the proapoptotic effect of chelerythrine in hESC-CM demonstrating an inhibitory role of calcineurin/NFAT and mammalian target of rapamycin (mTOR) pathways in hESC-CM survival (using apoptotic marker caspase-3), whereas the protection was less obvious in hESC-EC exposed to H2O2. Immunosuppressants did not affect cell viability in hESC-EC. Our results show that immunosuppressants reduce proliferation, while offsetting cell loss to a smaller extent by reduction in apoptosis of hESC-CM. Immunosuppressant therapy would be compatible with stem cell transplantation, but the resulting reduction in graft expansion capabilities would potentially necessitate implantation of increased cell numbers when immunosuppressants are given. The effects of NFAT-binding immunosuppressant molecules, which do not affect hESC-CM proliferation, may point the way forward for new classes of compounds better suited to cell implantation.

The effect of partial food deprivation on the astroglia in the dorsal subnucleus of the lateral septum of the rat brain.

The effect of 40% partial food deprivation was studied on the immunohistochemically detectable amount of glial fibrillary acidic protein (GFAP) - the specific marker of astroglia - in the dorsal subnucleus of lateral septum (LS) of male, intact and ovariectomized (OVX) female rats. Animals were either fed ad libitum (control) or 40% food deprived for one week, then perfusion-fixed, their brains removed, and serial vibratome sections were processed for the immunocytochemical localization of GFAP. Computeraided densitometry was carried out on digital photographs.The results showed that ovariectomy alone did not exert any effect on the density of GFAPimmunoreactivity (GFAP-IR) as compared to the values detected in intact females. Food deprivation increased the density of GFAP in each experimental group. The difference was most pronounced in males, significant in females and much less in ovariectomized females. Parietal cortex chosen as reference area did not show any increase in the local GFAP-IR.It was previously shown that the dorsal subnucleus of the lateral septum reacts with plastic neurochemical changes to food deprivation. Our results prove that these changes affect not only neuronal but also glial elements.

Radiofrequency ablation at low irrigation flow rates using a novel 12-hole gold open-irrigation catheter.

BACKGROUND: High irrigation rates during radiofrequency (RF) ablation may cause fluid overload and limit lesion size. This in vivo animal study assessed the safety and efficacy of RF ablation at low irrigation rates using a novel 12-hole gold catheter. METHODS: A total of 103 lesions, created on the thigh of five mongrel dogs, were analyzed. Lesions were created using a 12-hole irrigated gold-tip (Au) and a six-hole irrigated platinum-iridium (PtIr) catheter (both 7F/3.5-mm electrode; BIOTRONIK SE & CO, KG, Berlin, Germany) in parallel and perpendicular orientation. RF current was delivered for 60 seconds at 30 W using 8 mL/min and 15 mL/min irrigation. Electrode temperature, steam pops, lesion dimensions, and coagulum formation were recorded. RESULTS: Electrode temperatures were lower for Au compared to PtIr in parallel (8 mL/min: 38.1 ± 1.7°C vs 48.0 ± 4.8°C, P < 0.0001; 15 mL/min: 36.0 ± 1.5°C vs 46.9 ± 5.4°C, P < 0.0001) and perpendicular position (15 mL/min: 35.5 ± 1.2°C vs 38.4 ± 2.5°C, P = 0.003). The number of steam pops between Au and PtIr was comparable for parallel (8 mL/min: 14% vs 27%, P = 0.65; 15 mL/min: 14% vs 43%, P = 0.21) and perpendicular orientation (8 mL/min: 25% vs 17%, P = 1.00; 15 mL/min: 18% vs 0%, P = 0.48). Au created larger volumes than PtIr at 8 mL/min irrigation (861 ± 251 mm(3) vs 504 ± 212 mm(3) , P = 0.004); however, for 15 mL/min, volumes were comparable (624 ± 269 mm(3) vs 768 ± 466 mm(3) , P = 0.46). No coagulum formation was observed for any of the catheters on the surface and catheter tip. CONCLUSION: RF ablation at low flow rate using a novel 12-hole irrigation Au catheter is safe and results in larger lesions than with a PtIr electrode.

Cocaine- and amphetamine-regulated transcript (CART) peptide-immunopositive neuronal elements in the lateral septum: rostrocaudal distribution in the male rat.

The morphological features and distribution of cocaine- and amphetamine-regulated transcript peptide immunoreactivity (CART-IR) were studied in the lateral septum (LS) of male rats using light and electron microscopic immunocytochemistry and computer-aided densitometry. CART-IR was detected along the rostrocaudal axis of the LS in varicose axonal fibers only, although immunoreactive cell bodies and dendrites were not detected. Pericellular basket-like arrangements around immunonegative cell bodies were present. From among the targets of such pericellular baskets, glutamic acid decarboxylase (GAD)-immunopositive and NPY-immunoreactive somata were identified. Thin varicose axons were present in each section, whereas thick varicose axons were restricted to the sections of rostral position only. CART-IR was observed in varicose fibers forming a dense subependymal plexus, from which solitary varicose fibers entered the ependymal layer. The fine structure of varicosities was similar to that of other neuropeptide-containing fibers. Small varicosities established asymmetrical synaptic contacts mainly with dendrites and dendritic spines, and larger varicosities established symmetrical synapses with somata and dendritic shafts. CART-to-CART connections were not revealed. The density curve of the CART-IR along the rostrocaudal axis of LS was found to be paraboloid. CART is known as one of the most anorexigenic peptides. These results serve as basis for further physiological studies concerning the biological significance of lateral septal CART peptide in the regulation of food intake.

Innate immunity in human embryonic stem cells: comparison with adult human endothelial cells.

Treatment of human disease with human embryonic stem cell (hESC)-derived cells is now close to reality, but little is known of their responses to physiological and pathological insult. The ability of cells to respond via activation of Toll like receptors (TLR) is critical in innate immune sensing in most tissues, but also extends to more general danger sensing, e.g. of oxidative stress, in cardiomyocytes. We used biomarker release and gene-array analysis to compare responses in hESC before and after differentiation, and to those in primary human endothelial cells. The presence of cardiomyocytes and endothelial cells was confirmed in differentiated cultures by immunostaining, FACS-sorting and, for cardiomyocytes, beating activity. Undifferentiated hESC did not respond with CXCL8 release to Gram positive or Gram negative bacteria, or a range of PAMPs (pathogen associated molecular patterns) for TLRs 1-9 (apart from flagellin, an activator of TLR5). Surprisingly, lack of TLR-dependent responses was maintained over 4 months of differentiation of hESC, in cultures which included cardiomyocytes and endothelial cells. In contrast, primary cultures of human aortic endothelial cells (HAEC) demonstrated responses to a broad range of PAMPs. Expression of downstream TLR signalling pathways was demonstrated in hESC, and IL-1beta, TNFalpha and INFgamma, which bypass the TLRs, stimulated CXCL8 release. NFkappaB pathway expression was also present in hESC and NFkappaB was able to translocate to the nucleus. Low expression levels of TLRs were detected in hESC, especially TLRs 1 and 4, explaining the lack of response of hESC to the main TLR signals. TLR5 levels were similar between differentiated hESC and HAEC, and siRNA knockdown of TLR5 abolished the response to flagellin. These findings have potential implications for survival and function of grafted hESC-derived cells.