Nitric Oxide Inhibits Vascular Smooth Muscle Cell Proliferation and Neointimal Hyperplasia by Increasing the Ubiquitination and Degradation of UbcH10

Nitric oxide (NO) limits formation of neointimal hyperplasia in animal models of arterial injury in large part by inhibiting vascular smooth muscle cell (VSMC) proliferation through cell cycle arrest. The ubiquitin-conjugating enzyme UbcH10 is responsible for ubiquitinating cell cycle proteins for proper exit from mitosis. We hypothesize that NO prevents VSMC proliferation, and hence neointimal hyperplasia, by decreasing levels of UbcH10. Western blotting and immunofluorescent staining showed that NO reduced UbcH10 levels in a concentration-dependent manner in VSMC harvested from the abdominal aortas of Sprague-Dawley rats. Treatment with NO or siRNA to UbcH10 decreased both UbcH10 levels and VSMC proliferation (P<0.001), while increasing UbcH10 levels by plasmid transfection or angiotensin II stimulation increased VSMC proliferation to 150% (P=0.008) and 212% (P=0.002) of control, respectively. Immunofluorescent staining of balloon-injured rat carotid arteries showed a ~4-fold increase in UbcH10 levels, which was profoundly decreased following treatment with NO. Western blotting of carotid artery lysates showed no UbcH10 in uninjured vessels, a substantial increase in the injury alone group, and a significant decrease in the injury+NO group (~3-fold reduction versus injury alone). Importantly, in vitro and in vivo, a marked increase in polyubiquitinated UbcH10 was observed in the NO-treated VSMC and carotid arteries, respectively, indicating that NO may be decreasing unmodified UbcH10 levels by increasing its ubiquitination. Central to our hypothesis, we report that NO decreases UbcH10 levels in VSMC in vitro and following arterial injury in vivo in association with increasing polyubiquitinated-UbcH10 levels. These changes in UbcH10 levels correlate with VSMC proliferation and neointimal hyperplasia, making UbcH10 a promising therapeutic target for inhibiting this proliferative disease.     

Provision of nestboxes raises the breeding density of Great Tits Parus major equally in coniferous and deciduous woodland

Nestbox provision is a technique used to increase nest-site availability for secondary cavity-nesting birds. However, little is known about the demographic consequences of nestbox provision in different habitat types. To assess how nestbox provision affects the density of hole-nesting birds simultaneously in two contrasting habitats, we compared the breeding density of Great Tits along transects without nestboxes with that in transects where nestboxes were provided. Although the initial density of breeders was considerably higher in the deciduous habitat than in the coniferous habitat, provision of nestboxes increased density by a similar number of additional pairs in each habitat type. Thus, the provision of nestboxes in managed coniferous forests may be as effective in increasing the breeding opportunities of cavity nesters as in deciduous stands. Moreover, previous research showed that pairs in deciduous habitat with nestboxes have consistently lower breeding success than those in coniferous habitat with nestboxes. It is possible that the addition of nestboxes in the preferred habitat increased density to such an extent that density-dependent effects became apparent.     

Nitric oxide decreases activity and levels of the 11S proteasome activator PA28 in the vasculature

The 11S proteasome activator (PA28) binds to the 20S proteasome and increases its ability to degrade small peptides. Expression of PA28 subunits (α, β, γ) is induced by interferon-γ stimulation. Inflammation plays a role in the development of neointimal hyperplasia, and we have previously shown that nitric oxide (NO) reduces neointimal hyperplasia in animal models and 26S proteasome activity in rat aortic smooth muscle cells (RASMC). Here, we show that PA28 increased 26S proteasome activity in RASMC, as measured by a fluorogenic assay, and the NO donor S-nitroso N-acetylpenicillamine significantly inhibits this activation. This effect was abrogated by the reducing agents dithiothreitol and HgCl(2), suggesting that NO affects the activity of PA28 through S-nitrosylation. NO did not appear to affect PA28 levels or intracellular localization in RASMC in vitro. Three days following rat carotid artery balloon injury, levels of PA28α, β and γ subunits were decreased compared to uninjured control arteries (n=3/group) in vivo. The NO donor proline NONOate further decreased PA28α, β and γ levels by 1.9-, 2.3- and 3.4-fold, respectively, compared to uninjured control arteries. Fourteen days following arterial injury, levels of PA28α, β and γ subunits were increased throughout the arterial wall compared to uninjured control arteries, but were greatest for the α and β subunits. NO continued to decrease the levels of all three PA28 subunits throughout the arterial wall at this time point. Since the PA28 subunits are involved in the breakdown of peptides during inflammation, PA28 inhibition may be one mechanism by which NO inhibits neointimal hyperplasia.     

The European Renal Genome Project: An Integrated Approach Towards Understanding the Genetics of Kidney Development and Disease

Rapid progress in genome research creates a wealth of information on the functional annotation of mammalian genome sequences. However, as we accumulate large amounts of scientific information we are facing problems of how to integrate and relate the data produced by various genomic approaches. Here, we propose the novel concept of an organ atlas where diverse data from expression maps to histological findings to mutant phenotypes can be queried, compared and visualized in the context of a three-dimensional reconstruction of the organ. We will seek proof of concept for the organ atlas by elucidating genetic pathways involved in development and pathophysiology of the kidney. Such a kidney atlas may provide a paradigm for a new systems-biology approach in functional genome research aimed at understanding the genetic bases of organ development, physiology and disease.Key Words: EuReGene, kidney, genome, development, pathophysiology, genetics     

Heightened efficacy of nitric oxide-based therapies in type II diabetes mellitus and metabolic syndrome

Type II diabetes mellitus (DM) and metabolic syndrome are associated with accelerated restenosis following vascular interventions due to neointimal hyperplasia. The efficacy of nitric oxide (NO)-based therapies is unknown in these environments. Therefore, the aim of this study is to examine the efficacy of NO in preventing neointimal hyperplasia in animal models of type II DM and metabolic syndrome and examine possible mechanisms for differences in outcomes. Aortic vascular smooth muscle cells (VSMC) were harvested from rodent models of type II DM (Zucker diabetic fatty), metabolic syndrome (obese Zucker), and their genetic control (lean Zucker). Interestingly, NO inhibited proliferation and induced G0/G1 cell cycle arrest to the greatest extent in VSMC from rodent models of metabolic syndrome and type II DM compared with controls. This heightened efficacy was associated with increased expression of cyclin-dependent kinase inhibitor p21, but not p27. Using the rat carotid artery injury model to assess the efficacy of NO in vivo, we found that the NO donor PROLI/NO inhibited neointimal hyperplasia to the greatest extent in type II DM rodents, followed by metabolic syndrome, then controls. Increased neointimal hyperplasia correlated with increased reactive oxygen species (ROS) production, as demonstrated by dihydroethidium staining, and NO inhibited this increase most in metabolic syndrome and DM. In conclusion, NO was surprisingly a more effective inhibitor of neointimal hyperplasia following arterial injury in type II DM and metabolic syndrome vs. control. This heightened efficacy may be secondary to greater inhibition of VSMC proliferation through cell cycle arrest and regulation of ROS expression, in addition to other possible unidentified mechanisms that deserve further exploration.