Identifying gaps in disease knowledge among patients with peripheral artery disease.

OBJECTIVE: An individual's understanding of disease risk factors and outcomes is important for the ability to make healthy lifestyle choices and decisions about disease treatment. Peripheral artery disease (PAD) is a condition with increasing global prevalence and high risk of adverse patient outcomes. This study seeks to understand the adequacy of disease understanding in patients with PAD. METHODS: This was an observational study of patients with PAD recruited from vascular surgery outpatient clinic and PAD clinical studies at a single academic medical center over an 8-month period. A 44-item paper survey assessed demographic and socioeconomic information, knowledge of personal medical history, PAD risk factors, consequences of PAD, and health education preferences. Patients with documented presence of PAD were offered the survey. Patients unable to complete the survey or provide informed consent were not considered eligible. Disease "awareness" was defined as correct acknowledgement of the presence or absence of a disease, including PAD, in the personal medical history. "PAD knowledge score" was the percentage of correct responses to questions on general PAD risk factors and consequences. Of 126 eligible patients, 109 participated. Bivariate analysis was used to study factors associated with awareness of PAD diagnosis. Factors associated with the PAD knowledge score were studied using the Pearson correlation coefficient, two-sample t test, or one-way analysis of variance. P value < .05 was considered statistically significant. RESULTS: The mean participant age was 69.4 ± 11.0 years, and 39.4% (n = 43) were female. Most participants (78.9%; n = 86) had critical limb-threatening ischemia. Only 65.4% (n = 70) of participants were aware of a diagnosis of PAD, which was less than their awareness of related comorbidities. Factors positively associated with PAD diagnosis awareness were female sex (81.4% vs 54.7%; P = .004) and history of percutaneous leg revascularization (78.6% vs 47.9%; P = .001). Among 17 patients who had undergone major leg amputation, 35% (n = 6) were unaware of a diagnosis of PAD. PAD knowledge scores correlated positively with an awareness of PAD diagnosis (59.1% vs 48.7%; P = .02) and negatively with a history of hypertension (53.4% vs 68.1%; P = .001). Most participants (86.5%; n = 90) expressed a desire to be further educated on PAD. The most popular education topics were dietary recommendations, causes, and treatment for PAD. CONCLUSIONS: Patients with PAD have deficits in their awareness of this diagnosis and general knowledge about PAD. Future research priorities should further define these deficits and their causes in order to inform new strategies that foster information-seeking behavior and effective educational programs for PAD.

Current status of patient-reported outcome measures in vascular surgery.

A previously published review focused on generic and disease-specific patient-reported outcome measures (PROMs) relevant to vascular surgery but limited to arterial conditions. The objective of this project was to identify all available PROMs relevant to diseases treated by vascular surgeons and to evaluate vascular surgeon perceptions, barriers to widespread implementation, and concerns regarding PROMs. We provide an overview of what a PROM is and how they are developed, and summarize currently available PROMs specific to vascular surgeons. We also report results from a survey of 78 Society for Vascular Surgery members serving on committees within the Policy and Advocacy Council addressing the barriers and facilitators to using PROMs in clinical practice. Finally, we report the qualitative results of two focus groups conducted to assess granular perceptions of PROMS and preparedness of vascular surgeons for widespread implementation of PROMs. These focus groups identified a lack of awareness of existing PROMs, knowledge of how PROMs are developed and validated, and clarity around how PROMs should be used by the clinician as main subthemes for barriers to PROM implementation in clinical practice.

Why Temporary Filters Are Not Removed: Clinical Predictors in 1,000 Consecutive Cases.

BACKGROUND: Compared to permanent inferior vena cava (IVC) filters, higher complication rates occur with long-term use of temporary IVC filters. We aimed to identify patient clinical factors at the time of placement that could predict failure to remove a temporary IVC filter. METHODS: A retrospective review was performed of both vascular surgery and interventional radiology prospective databases between December 2008 and December 2013. We analyzed a total number of 1,024 consecutive, temporary IVC filters stratified by whether retrieval was attempted or made permanent. Univariate, multivariate, and prediction modeling analyses with internal validation were performed on abstracted data, which included risk factors, treatment modalities, and indications for IVC filter placement. RESULTS: Of 1,024 temporary IVC filters, removal was attempted in 60% and no attempt at removal (kept permanent) in 40%. Of the 619 with attempted removal, the overall successful retrieval rate was 95%. The majority of filters were not attempted to be removed because of persistent filter indications (360 cases). Risk factors associated with IVC filter permanence included male sex, older age, history, or indication of venous thromboembolism (VTE) with inability to anticoagulate, malignancy, and neurologic condition. Risk factors most predictive of permanence in the multivariate model were malignancy (odds ratio [OR]: 3.0, P < 0.001) or neurologic disorder (OR: 2.69, P = 0.0005). Validation revealed our model had a sensitivity of 60.4% and specificity of 69.9%. CONCLUSIONS: Our study shows that patients who are more likely to have a temporary IVC filter kept permanent are more likely to be older males with a history of malignancy, neurologic condition, or VTE. These factors are also predictive of permanence and can be used in our predictive model to provide insight into the significant preoperative risk factors that should play into the decision-making process.

Treatment options for symptomatic carotid stenosis: timing and approach.

Stroke is a major cause of morbidity and mortality and up to 15-20% of ischemic strokes can be attributed to atherosclerotic internal carotid artery disease. The treatment of carotid artery disease has been the subject of a wealth of literature in the past twenty years since the publication of the landmark randomized controlled trials, the North American Symptomatic Carotid Endarterectomy Trial and the European Carotid Surgery Trial, in the early 1990s. Although these landmark trials have helped establish the current guidelines for treatment of patients with symptomatic carotid artery disease, there have since been major advancements in the medical treatment of cardiovascular disease and there still remains a great deal of controversy regarding the timing and technical approach to carotid revascularization. In particular, there has been a wealth of literature to determine whether carotid endarterectomy or carotid stenting should be used for revascularization and when this revascularization should occur following onset of symptoms. This update offers an overview of the standards for diagnosis and medical treatment of patients with symptomatic carotid artery disease, the indications for surgical revascularization and a review of the most pertinent literature as it pertains to the more controversial issues of technical approach and timing of surgical revascularization following onset of symptoms in patients with carotid artery disease.

Debate: Whether evidence supports reducing the threshold diameter to 5 cm for elective interventions in women with abdominal aortic aneurysms.

Current practice guidelines recommend repair of asymptomatic abdominal aortic aneurysms once they reach the 5.5-cm-diameter threshold and are based on information from randomized controlled trials. However, because aneurysms are more common in men, women are under-represented in these trials, and questions persist about 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 anatomic candidates for endovascular repair, and do poorer after emergency 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.

Risk Factors for Unplanned Higher-Level Re-Amputation and Mortality after Lower Extremity Amputation in Chronic Limb-Threatening Ischemia.

Background: The factors associated with unplanned higher-level re-amputation (UHRA) and one-year mortality among patients with chronic limb-threatening ischemia (CLTI) after lower extremity amputation are poorly understood. Methods: This was a single-center retrospective study of patients who underwent amputations for CLTI between 2014 and 2017. Unadjusted bivariate analyses and adjusted odds ratios (AOR) from logistic regression models were used to assess associations between pre-amputation risk factors and outcomes (UHRA and one-year mortality). Results: We obtained data on 203 amputations from 182 patients (median age 65 years [interquartile range (IQR) 57, 75]; 70.7% males), including 118 (58.1%) toe, 20 (9.9%) transmetatarsal (TMA), 37 (18.2%) below-knee (BKA), and 28 (13.8%) amputations at or above the knee. Median follow-up was 285 days (IQR 62, 1348). Thirty-six limbs (17.7%) had a UHRA, and the majority of these (72.2%) were following index forefoot amputations. Risk factors for UHRA included non-ambulatory status (AOR 6.74, 95% confidence interval (CI) 1.74-26.18; p < 0.10) and toe pressure < 30 mm Hg (AOR 4.89, 95% CI 1.52-15.78; p < 0.01). One-year mortality was 17.2% (n = 32), and risk factors included coronary artery disease (AOR 3.93, 95% CI 1.56-9.87; p < 0.05), congestive heart failure (AOR 4.90, 95% CI 1.96-12.29; p = 0.001), end-stage renal disease (AOR 7.54, 95% CI 3.10-18.34; p < 0.001), and non-independent ambulation (AOR 4.31, 95% CI 1.20-15.49; p = 0.03). Male sex was associated with a reduced odds of death at 1 year (AOR 0.37, 95% CI 0.15-0.89; p < 0.05). UHRA was not associated with one-year mortality. Conclusions: Rates of UHRA after toe amputations and TMA are high despite revascularization and one-year mortality is high among patients with CLTI requiring amputation.

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.

Nitric oxide may inhibit neointimal hyperplasia by decreasing isopeptidase T levels and activity in the vasculature.

OBJECTIVE: Isopeptidase T is a cysteine protease deubiquitinating enzyme that hydrolyzes unanchored polyubiquitin chains to free monoubiquitin. Nitric oxide (NO) decreases 26S proteasome activity in vascular smooth muscle cells (VSMCs) and inhibits neointimal hyperplasia in animal models. As NO can cause S-nitrosylation of active-site cysteines, we hypothesize that NO inhibits isopeptidase T activity through S-nitrosylation. Because accumulation of polyubiquitin chains inhibits the 26S proteasome, this may be one mechanism through which NO prevents neointimal hyperplasia. METHODS: To investigate our hypothesis, we examined the effect of NO on isopeptidase T activity, levels, and localization in VSMCs in vitro and in a rat carotid balloon injury model in vivo. RESULTS: NO inhibited recombinant isopeptidase T activity by 82.8% (t = 60 minutes, P < .001 vs control). Dithiothreitol and glutathione (5 mmol/L) both significantly reversed NO-mediated inhibition of isopeptidase T activity (P < .001). NO caused a time-dependent increase in S-nitrosylated isopeptidase T levels in VSMCs, which was reversible with dithiothreitol, indicating that isopeptidase T undergoes reversible S-nitrosylation on exposure to NO in vitro. Although NO did not affect isopeptidase T levels or subcellular localization in VSMCs in vitro, it decreased isopeptidase T levels and increased ubiquitinated proteins after balloon injury in vivo. CONCLUSIONS: Local administration of NO may prevent neointimal hyperplasia by inhibiting isopeptidase T levels and activity in the vasculature, thereby inhibiting the 26S proteasome in VSMCs. These data provide additional mechanistic insights into the ability of NO to prevent neointimal hyperplasia after vascular interventions.

Nitric oxide is less effective at inhibiting neointimal hyperplasia in spontaneously hypertensive rats.

Exogenous administration of nitric oxide (NO) markedly decreases neointimal hyperplasia following arterial injury in several animal models. However, the effect of NO on neointimal hyperplasia in hypertension remains unknown. Here, we employ the spontaneously hypertensive rat (SHR) strain, inbred from Wistar Kyoto (WKY) rats, and the carotid artery balloon injury model to assess the effects of NO on neointimal hyperplasia development. 2weeks after arterial injury, we showed that both rat strains developed similar levels of neointimal hyperplasia, but local administration of NO was less effective at inhibiting neointimal hyperplasia in the SHR compared to WKY rats (58% vs. 79%, P<0.001). Interestingly, local administration of NO did not affect systemic blood pressure in either rat strain. Compared to WKY, the SHR displayed more proliferation in the media and adventitia following balloon injury, as measured by BrdU incorporation. The SHR also showed more inflammation in the adventitia after injury, as well as more vasa vasorum, than WKY rats. NO treatment reduced the vasa vasorum in the SHR but not WKY rats. Finally, while NO decreased both injury-induced proliferation and inflammation in the SHR, it did not return these parameters to levels seen in WKY rats. We conclude that NO is less effective at inhibiting neointimal hyperplasia in the SHR than WKY rats. This may be due to increased scavenging of NO in the SHR, leading to diminished bioavailability of NO. These data will help to develop novel NO-based therapies that will be equally effective in both normotensive and hypertensive patient populations.

Open aortic septectomy for complicated type B aortic dissection.

Malperfusion is a complication of acute aortic dissection associated with substantially increased morbidity and mortality. Although endovascular treatment of the dissection with a stent graft to cover the intimal tear and reexpand the true lumen will often be sufficient to treat distal malperfusion, persistent or delayed malperfusion will necessitate additional interventions. Endovascular strategies to increase true lumen expansion include bare metal dissection stent placement and percutaneous fenestration. However, for patients with anatomy not amenable to an endovascular approach, alternative techniques are required. We describe two cases of complicated acute aortic dissection due to partial false lumen thrombosis treated with open aortic septectomy. Although an uncommon procedure, open septectomy can be useful for patients with malperfusion syndromes without appropriate endovascular options.

Efficacy and safety of sonographer discretion to terminate a venous duplex ultrasound for diagnosis of deep vein thrombosis in coronavirus disease 2019 patients.

OBJECTIVE: Sonographers performing venous duplex ultrasound (VDUS) of patients with coronavirus disease 2019 (COVID-19) have an increased risk of exposure owing to their close contact with these patients for an extended period. The objective of the present study was to evaluate the efficacy of a modified COVID-19 VDUS protocol to reduce sonographer exposure to COVID-19 patients. METHODS: We performed a single-center retrospective review. Patients who had undergone VDUS under the modified COVID-19 protocol between March 1, 2020, and June 30, 2020, with a confirmed or presumed COVID-19 diagnosis at the VDUS were included. The modified COVID-19 protocol was defined as the ability of the sonographer to terminate the examination on detection of an acute deep vein thrombosis (DVT). The primary outcome measures were the number of anatomic deep venous segments recorded by the sonographer, which was used as a surrogate measure for sonographer exposure time, and the number of acute DVTs found on follow-up examinations in segments not visualized at the index VDUS. RESULTS: A total of 160 lower extremity VDUS (LEVDUS) scans and 72 upper extremity VDUS (UEVDUS) scans were performed using the modified COVID-19 protocol. The index VDUS had found an acute DVT for 44 of 160 patients (27.5%) who had undergone LEVDUS and 26 of 72 (36.6%) who had undergone UEVDUS. On follow-up imaging, 7 of 38 LEVDUS scans (17.9%) and 1 of 10 UEVDUS scans (10%) had demonstrated a new acute DVT. Malignancy and surgery 30 days before imaging were significantly associated with acute lower extremity DVT, and mechanical ventilation and extracorporeal membrane oxygenation were associated with acute upper extremity DVT. On the index VDUS, the average was 10.6 of 12 total visualized segments on LEVDUS and 6.4 of 10 total segments on UEVDUS. Of the index VDUS scans, 35.6% of the LEVDUS and 78.6% of the UEVDUS scans had been abbreviated. The index VDUS scans that were positive for acute DVT had had significantly fewer visualized segments for both lower (8.4 vs 11.5; P < .0001) and upper (4.2 vs 7.6) extremities (P < .0001). On the follow-up examinations, only one of eight new acute DVTs had been found in a patient whose index VDUS had been abbreviated and the corresponding segment not assessed. These findings did not affect the patient's clinical course. CONCLUSIONS: The modified COVID-19 VDUS protocol reduced sonographers' potential exposure time to COVID-19. Additionally, the clinical efficacy was maintained, with no missed DVTs, despite the abbreviation of the VDUS examinations.

Unconscious bias in speaker introductions at a national vascular surgery meeting: The impact of rank, race and gender.

OBJECTIVES: Unconscious bias can impact manner of speaker introductions in formal academic settings. We examined speaker introductions at the Society of Vascular Surgeons Annual Meeting to determine factors associated with non-professional address. METHODS: We examined speaker introductions from the 2019 SVS Vascular Annual Meeting. Professional title with either full name or last name was considered professional address. Speaker and moderator demographics were collected. Univariate and multivariate logistic regression analyses were performed to identify associations between introduction and speaker and moderator characteristics. RESULTS: 336 talks met inclusion criteria. Both speakers and moderators were more likely to be white (63.4 â€‹% and 65.8 â€‹%,p â€‹= â€‹0.92), man (75.6 â€‹% and 74.4 â€‹%,p â€‹= â€‹0.82) and full professor rank (34.5 â€‹% and 42.3 â€‹%, p â€‹< â€‹0.001). On multivariable regression, non-professional address was associated with speaker rank of trainee (OR 3.13, p â€‹= â€‹0.05) and when moderator was white (OR 2.42, p â€‹= â€‹0.03). CONCLUSIONS: This study emphasizes the potential negative impact of unconscious bias at a national meeting for vascular surgeons and the need to mitigate this effect at the organization level.

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, ß, 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, ß and γ subunits were decreased compared to uninjured control arteries (n=3/group) in vivo. The NO donor proline NONOate further decreased PA28α, ß 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, ß and γ subunits were increased throughout the arterial wall compared to uninjured control arteries, but were greatest for the α and ß 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 role of estrogen receptor α and ß 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 (ß) inhibitor (R,R)-5,11-Diethyl-5,6,11,12-tetrahydro-2,8-chrysenediol, the combined ERαß 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 ERß knockout (ERß KO) mice in the presence or absence of DETA/NO and the ERα, ERß, and ERαß inhibitors. Protein levels were assessed using Western blot analysis. RESULTS: Protein expression of ERα and ERß was present and equal in male and female VSMC, and did not change after exposure to NO. Inhibition of either ERα or ERß had no effect on VSMC proliferation in the presence or absence of NO in either sex. However, inhibition of ERαß 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 ERßKO VSMC proliferated faster than male WT VSMC (P < 0.05). Female ERßKO 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 ERß in these knockout strains. Combined ERαß 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.

Application of dissemination and implementation science frameworks to surgical research.

A statistic often quoted is that <15% of research is translated into practice and routine adoption of novel research findings can take up to 17 years. Dissemination and implementation science is a field of research focused on studying and developing approaches that can increase the uptake of such innovations, thereby reducing the significant time lag between scientific discovery and widespread adoption. As such, it can have a major clinical impact by increasing the utilization of proven innovations in routine clinical practice using systematic frameworks to implement, disseminate, and evaluate the successful application of evidence-based practices. Herein, we discuss the background and theory of implementation science, major frameworks and considerations for study design, and current examples of its application in surgical research.

Characterizing endovascular aortic intervention outcomes for nonruptured aortic aneurysms by physician specialty.

BACKGROUND: Evaluate patient outcomes after endovascular aortic interventions performed for nonruptured aortic aneurysms by physician specialties. METHODS: Endovascular aortic repair (EVAR), fenestrated or branched repair (F-BEVAR), and thoracic endovascular aortic repair (TEVAR) procedures were obtained from the Illinois Hospital Association Comparative Health Care and Hospital Data Reporting Services database from 2016 to 2019. Logistic and Poisson regression were used to determine outcomes by patient, physician, and hospital characteristics. RESULTS: A total of 4,935 procedures, 3,666 (74.3%) EVAR, 567 (11.5%) F-BEVAR, and 702 (14.2%) TEVAR were performed by vascular surgeons, interventional radiologists, interventional cardiologists, and cardiac surgeons. Vascular surgeons performed interventions equally between hospital types while interventional radiologists primarily performed interventions in teaching hospitals (68.1%) and interventional cardiologists and cardiac surgeons typically performed interventions in community hospitals (91.8% and 82.1%, respectively; P < .001). No differences in inpatient mortality were noted between specialties. Patients treated by interventional radiologists had increased odds of staying in the hospital ≥8 days (odd ration [OR] 1.95, 95% confidence interval [CI] 1.19-3.19) and patients treated by interventional cardiologists had lower odds of being admitted to the intensive care unit [ICU] (OR 0.42, 95% CI 0.18-0.95). CONCLUSION: Differences in practice patterns among specialties performing endovascular aortic aneurysm repair for nonruptured aneurysms suggest opportunities for collaboration to optimize quality of care.

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.

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.

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.

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.