Ashley K. Vavra

Regulation of reactive oxygen species by p53: implications for nitric oxide-mediated apoptosis

Nitric oxide (NO) induces vascular smooth muscle cell (VSMC) apoptosis in part through activation of p53. Traditionally, p53 has been thought of as the gatekeeper, determining if a cell should undergo arrest and repair or apoptosis following exposure to DNA-damaging agents, depending on the severity of the damage. However, our laboratory previously demonstrated that NO induces apoptosis to a much greater extent in p53(-/-) compared with p53(+/+) VSMC. Increased reactive oxygen species (ROS) within VSMC has been shown to induce VSMC apoptosis, and recently it was found that the absence of, or lack of, functional p53 leads to increased ROS and oxidative stress within different cell types. This study investigated the differences in intracellular ROS levels between p53(-/-) and p53(+/+) VSMC and examined if these differences were responsible for the increased susceptibility to NO-induced apoptosis observed in p53(-/-) VSMC. We found that p53 actually protects VSMC from NO-induced apoptosis by increasing antioxidant protein expression [i.e., peroxiredoxin-3 (PRx-3)], thereby reducing ROS levels and cellular oxidative stress. We also observed that the NO-induced apoptosis in p53(-/-) VSMC was largely abrogated by pretreatment with catalase. Furthermore, when the antioxidant protein PRx-3 and its specific electron acceptor thioredoxin-2 were silenced within p53(+/+) VSMC with small-interfering RNA, not only did these cells exhibit greater ROS production, but they also exhibited increased NO-induced apoptosis similar to that observed in p53(-/-) VSMC. These findings suggest that ROS mediate NO-induced VSMC apoptosis and that p53 protects VSMC from NO-induced apoptosis by decreasing intracellular ROS. This research demonstrates that p53 has antioxidant functions in stressed cells and also suggests that p53 has antiapoptotic properties.

Peripheral arterial disease in women

Peripheral arterial disease (PAD) affects a significant portion of the United States population, and much research has been conducted on identifying populations at risk for PAD, evaluating appropriate diagnostic modalities for PAD, studying the effect of risk factor reduction on PAD progression, and determining the best method of treatment for symptomatic PAD. However, most PAD research and clinical trials have focused on whole populations, or populations consisting mostly of men. Little data exist with respect to PAD in women. The goal of this review is to highlight what is known about gender-related differences for PAD.

Poly(diol‐co‐citrate)s as Novel Elastomeric Perivascular Wraps for the Reduction of Neointimal Hyperplasia

The synthesis of poly(diol-co-citrate) elastomers that are biocompatible with vascular cells and can modulate the kinetics of the NO release based on the diol of selection is reported. NO-mediated cytostatic or cytotoxic effects can be controlled depending on the NO dose and the exposure time. When implanted in vivo in a rat carotid artery injury model, these materials demonstrate a significant reduction of neointimal hyperplasia. This is the first report of a NO-releasing polymer fabricated in the form of an elastomeric perivascular wrap for the treatment of neointimal hyperplasia. These elastomers also show promise for other cardiovascular pathologies where NO-based therapies could be beneficial.

Effect of Nitric Oxide on Neointimal Hyperplasia based on Sex and Hormone Status

Nitric oxide (NO)-based therapies decrease neointimal hyperplasia; however, studies have been performed only in male animal models. Thus, we sought to evaluate the effect of NO on vascular smooth muscle cells (VSMC) in vitro and neointimal hyperplasia in vivo based on sex and hormone status. In hormone-replete medium, male VSMC proliferated at greater rates than female VSMC. In hormone-depleted medium, female VSMC proliferated at greater rates than male VSMC. However, in both hormone environments, NO inhibited proliferation and migration to a greater extent in male compared to female VSMC. These findings correlated with greater G₀/G₁ cell cycle arrest and changes in cell cycle protein expression in male compared to female VSMC after exposure to NO. Next, the rat carotid artery injury model was used to assess the effect of NO on neointimal hyperplasia in vivo. Consistent with the in vitro data, NO was significantly more effective at inhibiting neointimal hyperplasia in hormonally intact males compared to females using weight-based dosing. An increased weight-based dose of NO in females was able to achieve efficacy equal to that in males. Surprisingly, NO was less effective at inhibiting neointimal hyperplasia in castrated animals of both sexes. In conclusion, these data suggest that NO inhibits neointimal hyperplasia more effectively in males compared to females and in hormonally intact compared to castrated rats, indicating that the effects of NO in the vasculature may be sex- and hormone-dependent.

Insulin enhances the effect of nitric oxide at inhibiting neointimal hyperplasia in a rat model of type 1 diabetes

Diabetes confers greater restenosis from neointimal hyperplasia following vascular interventions. While localized administration of nitric oxide (NO) is known to inhibit neointimal hyperplasia, the effect of NO in type 1 diabetes is unknown. Thus the aim of this study was to determine the efficacy of NO following arterial injury, with and without exogenous insulin administration. Vascular smooth muscle cells (VSMC) from lean Zucker (LZ) rats were exposed to the NO donor, DETA/NO, following treatment with different glucose and/or insulin concentrations. DETA/NO inhibited VSMC proliferation in a concentration-dependent manner to a greater extent in VSMC exposed to normal-glucose vs. high-glucose environments, and even more effectively in normal-glucose/high-insulin and high-glucose/high-insulin environments. G(0)/G(1) cell cycle arrest and cell death were not responsible for the enhanced efficacy of NO in these environments. Next, type 1 diabetes was induced in LZ rats with streptozotocin. The rat carotid artery injury model was performed. Type 1 diabetic rats experienced no significant reduction in neointimal hyperplasia following arterial injury and treatment with the NO donor PROLI/NO. However, daily administration of insulin to type 1 diabetic rats restored the efficacy of NO at inhibiting neointimal hyperplasia (60% reduction, P < 0.05). In conclusion, these data demonstrate that NO is ineffective at inhibiting neointimal hyperplasia in an uncontrolled rat model of type 1 diabetes; however, insulin administration restores the efficacy of NO at inhibiting neointimal hyperplasia. Thus insulin may play a role in regulating the downstream beneficial effects of NO in the vasculature.

Insights into the effect of nitric oxide and its metabolites nitrite and nitrate at inhibiting neointimal hyperplasia

OBJECTIVE Periadventitial delivery of the nitric oxide (NO) donor PROLI/NO following arterial injury effectively inhibits neointimal hyperplasia. Given the short half-life of NO release from PROLI/NO, our goal was to determine if inhibition of neointimal hyperplasia by PROLI/NO was due to NO, or its metabolites nitrite and nitrate. METHODS AND RESULTS In vitro, the NO donor DETA/NO inhibited proliferation of rat aortic vascular smooth muscle cells (RASMC), but neither nitrite nor nitrate did. In vivo, following rat carotid artery balloon injury or injury plus the molar equivalents of PROLI/NO, nitrite, or nitrate (n=8-11/group), PROLI/NO was found to provide superior inhibition of neointimal hyperplasia (82% inhibition of intimal area, and 44% inhibition of medial area, p<0.001). Only modest inhibition was noted with nitrite or nitrate (45% and 41% inhibition of intimal area, and 31% and 29% inhibition of medial area, respectively, p<0.001). No effects on blood pressure were noted with any treatment groups. In vivo, only PROLI/NO inhibited cellular proliferation and increased arterial lumen area compared to injury alone (p<0.001). However, all three treatments inhibited inflammation (p<0.001). CONCLUSIONS PROLI/NO was more effective at inhibiting neointimal hyperplasia following arterial injury than nitrite or nitrate. However, modest inhibition of neointimal hyperplasia was observed with nitrite and nitrate, likely secondary to anti-inflammatory actions. In conclusion, we have demonstrated that the efficacy of NO donors is primarily due to NO production and not its metabolites, nitrite and nitrate.

The Role of Estrogen Receptor α and β in Regulating Vascular Smooth Muscle Cell Proliferation is Based on Sex

BACKGROUND We previously demonstrated that vascular smooth muscle cells (VSMC) proliferation and development of neointimal hyperplasia as well as the ability of nitric oxide (NO) to inhibit these processes is dependent on sex and hormone status. The aim of this study was to evaluate the role of estrogen receptor (ER) in mediating proliferation in male and female VSMC. MATERIALS AND METHODS Proliferation was assessed in primary rat aortic male and female VSMC using (3)H-thymidine incorporation in the presence or absence of ER alpha (α) inhibitor methyl-piperidino-pyrazole, the ER beta (β) inhibitor (R,R)-5,11-Diethyl-5,6,11,12-tetrahydro-2,8-chrysenediol, the combined ERαβ inhibitor ICI 182,780, and/or the NO donor DETA/NO. Proliferation was also assessed in primary aortic mouse VSMC harvested from wildtype (WT), ERα knockout (ERα KO), and ERβ knockout (ERβ KO) mice in the presence or absence of DETA/NO and the ERα, ERβ, and ERαβ inhibitors. Protein levels were assessed using Western blot analysis. RESULTS Protein expression of ERα and ERβ was present and equal in male and female VSMC, and did not change after exposure to NO. Inhibition of either ERα or ERβ had no effect on VSMC proliferation in the presence or absence of NO in either sex. However, inhibition of ERαβ in rat VSMC mitigated NO-mediated inhibition in female but not male VSMC (P < 0.05). Evaluation of proliferation in the knockout mice revealed distinct patterns. Male ERαKO and ERβKO VSMC proliferated faster than male WT VSMC (P < 0.05). Female ERβKO proliferated faster than female WT VSMC (P < 0.05), but female ERαKO VSMC proliferated slower than female WT VSMC (P < 0.05). Last, we evaluated the effect of combined inhibition of ERα and ERβ in these knockout strains. Combined ERαβ inhibition abrogated NO-mediated inhibition of VSMC proliferation in female WT and knockout VSMC (P < 0.05), but not in male VSMC. CONCLUSIONS These data clearly demonstrate a role for the ER in mediating VSMC proliferation in both sexes. However, these data suggest that the antiproliferative effects of NO may be regulated by the ER in females but not males.

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.

Part One: For the Motion. Evidence Supports Reducing the Threshold Diameter to 5 cm for Elective Interventions in Women With Abdominal Aortic Aneurysms

Current practice guidelines recommend the repair of asymptomatic abdominal aortic aneurysms once they reach the 5.5-cm diameter threshold, and are based on information from randomised controlled trials. However, as aneurysms are more common in men, women are under-represented in these trials, and questions persist with regard to whether this repair threshold should apply to them. In addition, women have smaller aortas to begin with and in most aneurysm cohorts are older, have more atherosclerotic risk factors, are less likely to be anatomical candidates for endovascular repair, and do less well after emergent or elective repair of their aneurysm. These are just some of the issues that our discussants address in determining whether the repair threshold should be at a smaller diameter for women.

Nitric oxide increases lysine 48-linked ubiquitination following arterial injury.

BACKGROUND Proteins are targeted for degradation by the addition of a polyubiquitin chain. Chains of ubiquitin linked via lysine 48 (K48) are associated with protein degradation while chains linked via lysine 63 (K63) are associated with intracellular signaling. We have previously shown that nitric oxide (NO) inhibits neointimal hyperplasia in association with increasing the ubiquitination and degradation of UbcH10. The aim of this study is to characterize the effect of arterial injury and NO on K48- or K63-linked ubiquitination of cellular proteins. METHODS The rat carotid artery balloon injury model was performed. Treatment groups included uninjured, injury alone, injury + proline NONOate (PROLI/NO), and PROLI/NO alone. Arteries were harvested at designated time points and sectioned for immunohistochemical analysis of K48- and K63-linked ubiquitination or homogenized for protein analysis. Vascular smooth muscle cells (VSMC) harvested from rat aortae were exposed to the NO donor diethylenetriamine NONOate (DETA/NO). Protein expression was determined by Western blot analysis, or immunoprecipitation and Western blot analysis. RESULTS Arterial injury increased K48-linked ubiquitination in vivo. The addition of PROLI/NO following injury caused a further increase in K48-linked ubiquitination at 1 and 3 d, however, levels returned to that of injury alone by 2 wk. Interestingly, treatment with PROLI/NO alone increased K48-linked ubiquitination in vivo to levels similar to injury alone. There were lesser or opposite changes in K63-linked ubiquitination in all three treatment groups. DETA/NO increased K48-linked ubiquitination in VSMC in vitro but had minimal effects on K63-linked ubiquitination. Low doses of DETA/NO decreased K48-linked ubiquitination of cyclin A and B, while high doses of DETA/NO increased K48-linked ubiquitination of cyclin A and B. Minimal changes were seen in K63-linked ubiquitination of cyclin A and B in vitro. CONCLUSIONS Arterial injury and NO increased K48-linked ubiquitination in vivo and in vitro. Remarkably, minimal changes were seen in K63-linked ubiquitination. These novel findings provide important insights into the vascular biology of arterial injury and suggest that one mechanism by which NO may prevent neointimal hyperplasia is through regulation of protein ubiquitination.