United States

Purpose:
Racial disparities in post-operative mortality following coronary artery bypass grafting (CABG) have been investigated, although underlying causes have yet to be elucidated. This study aims to analyze historical trends in 30-day postoperative
mortality rates between black and white patients, as well as the accuracy and methods used to elicit them.

Methods:
Studies published between 1984 and 2023 were collected from a nonstructured review in PubMed. Article inclusion criteria were patients of all ages who underwent CABG and explicit reporting of 30-day post-operative mortality in black and white patients. Studies that focused on non-CABG cardiac surgeries and studies that did not report 30-day mortality rates stratified by race were excluded. Studies meeting inclusion criteria were evaluated using title and abstract review. Subsequent full-text screening and data extraction were performed.

Results:
Differences in 30-day post-operative mortality rates still exist following CABG between black and white patients. (Image 1) Several studies have analyzed 30-day mortality rates stratified by race following CABG, however, there is a paucity of large database studies.(Table 1) Angraal et al. recently utilized Medicare data from the Centers for Medicare &amp; Medicaid Services (CMS) to report on CABG outcomes in patients over age 65 among different races between 1999-2014.(1) Between 2011 and 2020, the mean age of a patient undergoing primary isolated CABG was
66.0 years.(2) In our experience, 61.3% of patients who underwent CABG in the past five years at our academic inner-city hospital were below 65 years of age and exhibited a greater number of comorbid conditions compared with their older counterparts, and there may not be a database that accurately captures the scope of outcomes and disparities that exist in and between patients of different racial backgrounds.

Conclusion:
There is no standardized way to evaluate CABG outcomes between patients of different racial backgrounds, leading to potential discrepancies between true and reported 30-day mortality rates. However, previous studies show differences. Thus, a standardized system is necessary to accurately assess mortality between races and to define areas for quality improvement.

AMA Research Challenge 2024 

Racial Disparities in Mortality Rates Following Coronary Artery Bypass Grafting: An Evaluation of Reporting Methods and Trends

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AMA Research Challenge 2025 

Join the AMA Research Challenge poster symposium and semifinals to explore top research posters and score posters in the semifinals (an AMA member exclusive opportunity).

Purpose:
Racial disparities in post-operative mortality following coronary artery bypass grafting (CABG) have been investigated, although underlying causes have yet to be elucidated. This study aims to analyze historical trends in 30-day postoperative
mortality rates between black and white patients, as well as the accuracy and methods used to elicit them.

Methods:
Studies published between 1984 and 2023 were collected from a nonstructured review in PubMed. Article inclusion criteria were patients of all ages who underwent CABG and explicit reporting of 30-day post-operative mortality in black and white patients. Studies that focused on non-CABG cardiac surgeries and studies that did not report 30-day mortality rates stratified by race were excluded. Studies meeting inclusion criteria were evaluated using title and abstract review. Subsequent full-text screening and data extraction were performed.

Results:
Differences in 30-day post-operative mortality rates still exist following CABG between black and white patients. (Image 1) Several studies have analyzed 30-day mortality rates stratified by race following CABG, however, there is a paucity of large database studies.(Table 1) Angraal et al. recently utilized Medicare data from the Centers for Medicare & Medicaid Services (CMS) to report on CABG outcomes in patients over age 65 among different races between 1999-2014.(1) Between 2011 and 2020, the mean age of a patient undergoing primary isolated CABG was
66.0 years.(2) In our experience, 61.3% of patients who underwent CABG in the past five years at our academic inner-city hospital were below 65 years of age and exhibited a greater number of comorbid conditions compared with their older counterparts, and there may not be a database that accurately captures the scope of outcomes and disparities that exist in and between patients of different racial backgrounds.

Conclusion:
There is no standardized way to evaluate CABG outcomes between patients of different racial backgrounds, leading to potential discrepancies between true and reported 30-day mortality rates. However, previous studies show differences. Thus, a standardized system is necessary to accurately assess mortality between races and to define areas for quality improvement.

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### Grand Prize Sponsor

AMA would like to thank Laurel Road for sponsoring a $10,000 grand prize for the winner of the AMA Research Challenge. Laurel Road is not involved in winner selection process. The winner will be announced on February 20, 2025 during the AMA Research Challenge finals. Payment of the grand prize will be directly from Laurel Road. See the AMA Research Challenge Official Rules for terms and conditions.

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Background:

Lung adenocarcinoma is the most common lung cancer and has variable biology that affects outcomes and treatment. Numerous prognostic factors can be obtained from resected tumors, but a tool that could predict lung adenocarcinoma biology pre-operatively would help tailor decision-making. 

SILA is a radiomic tool that assigns scores to 9 groups of CT features which are collectively used to calculate a risk that a tumor recurs. While SILA recurrence risks have been validated, it is not yet known how the 9 groups of features themselves might relate to lung adenocarcinoma characteristics.

In this study, our aim was to elucidate novel associations between the 9 scores and known prognostic factors in lung adenocarcinoma. 

Methods:

We retrospectively reviewed resected lung adenocarcinoma cases with available preoperative CT images at our hospital between August 2016 and August 2022.

SILA analysis involved manual segmentation of lung lesions on CT scans. The software then generated scores for each of the 9 groups of features. The 9 features are referred to by color names, such as violet (V), indigo (I), etc. We reviewed the charts to record pertinent clinical-pathologic variables. 
Statistical tests used included chi square, Kruskal-Wallis to Dunn-Bonferroni-Tests post-hoc analysis, and Mann-Whitney U-Test.

Results:
Study cohort included 276 patients with a median age of 69. There were 176 (63.4%) female patients and 197 (71.4%) stage I. There were 54 (19.6%) recurrences.

Our results showed positive and negative associations between certain color scores and markers of tumor aggression, including standardized reuptake value (SUV) on T1 scans (p-values ranging from <0.001 to 0.009) and tumor size on CT (p-values from <0.001 to 0.009). In addition, color scores had associations with known prognostic factors that can only be determined after resection, including visceral pleural invasion (p-values from <0.001 to .045) and lymphovascular invasion (p values from <0.001 to .037).

Color scores were also significantly different between certain pathologic stages. For example, violet values were significantly different between stage IB and IA2 tumors (adjusted p=0.001) and between stage IA2 and IIB tumors (adjusted p<0.001). 

Conclusion:

The associations between SILA color scores and validated markers of tumor aggression point to the exciting possibility that color scores may provide information about lung adenocarcinoma biology pre-operatively. Future work will explore additional correlations between color scores and other pre- and post-operative clinicopathologic data.


Score Indicative of Lung Cancer Aggression (SILA) color scores are correlated with aggressive features in lung adenocarcinoma


Background
Compared to traditional laparoscopic radical nephrectomy (LRN), robotic surgery offers potential advantages such as improved surgical dexterity and enhanced anatomical visualization during surgery, which can improve dissection, tumor identification, and control of renal vessels, aspects critical for reconstructive surgery. It is unclear whether these potential benefits would translate to improved post-operative outcomes for patients undergoing radical nephrectomy; thus, our objective was to compare perioperative complications

Methods
We conducted a retrospective study using data from the American College of Surgeons-National Surgical Quality Improvement Program (ACS-NSQIP) Nephrectomy-Targeted database from 2019 to 2021, restricted to localized RCC. We utilized propensity score matching (1:1 PSM) to balance patient characteristics between the two groups. The association between the outcomes of interest (Primary Outcome as major complications within 30 days of surgery, including 30-day mortality, return to the operating room, cardiac arrest, MI, and CVA; Secondary outcome as other perioperative complications) and the operative modality was assessed using logistic regression.

Results
Among the 1,545 patients in the study (mean age: 62.9±11.8 years), 722 underwent RARN and 823 underwent LRN. We did not observe any differences in major complications [OR 0.93 (95% CI 0.43-2.00), p=0.848], including 30-day mortality, return rate to the operating room, cardiac arrest, myocardial infarction, and stroke or cerebrovascular accident between the operative modalities. LRN was associated with a 128% increased risk of surgical site infection [OR 2.28 (95% CI 1.01-5.16), p=0.047] and 54% increased risk of prolonged LOS (defined as >3 days) [OR 1.54 (95% CI 1.15-2.06), p=0.004), compared to RARN group. In addition, the conversion rate of LRN to open RN was 4.3%, while the conversion rate of RARN was 1.16% [OR 3.70 (3.25-4.15), p<0.001]. RARN was associated with significantly longer operative time (minutes) than LRN [Estimated coefficient: 30.67 (95% CI 23.15-38.18), p<0.001].

Conclusion
We found no significant difference in major complications between RARN and LRN for patients undergoing surgery for localized kidney cancer. At the expense of somewhat longer operative time, RARN was associated with a lower risk of SSI and lower conversion rate to open RN. These data support the notion that LRN and RARN should both be considered and selected on an individualized basis using tumor, patient, and physician factors. 




Comparison of Mortality and Morbidity of Robotic vs Laparoscopuc Radical Nephrectomy for the Treatment of Renal Cell Carcinoma – An Analysis of the National Surgery Quality Improvement Targeted Database

Abstract Title: Determining Best Treatments for Pediatric Bowel and Bladder Dysfunction

Introduction and objectives
Bowel and bladder dysfunction (BBD) describes a spectrum of symptoms including urinary and stool incontinence, urgency, frequency, painful urination, and constipation that accounts for over 40% of all pediatric urology clinic visits. Children with BBD may also suffer from recurrent UTIs, which can cause renal scarring and reduced kidney function. In addition to the urinary and gastrointestinal issues, BBD has a profound impact on a child’s mental health, self-esteem, and social development. Given the impact of these issues, it is important not only to properly diagnose BBD, but provide an effective treatment plan. Unfortunately, no defined criteria currently exist as to which therapies should be used by clinicians despite the wide range of treatment options including: urotherapy (timed voiding schedule), stool softeners, anticholinergics, desmopressin, electrical stimulation, surgical interventions, and pelvic floor physical therapy.

Methods
A retrospective chart review was performed to analyze questionnaire and treatment data from patients who presented to our pediatric urology clinic for evaluation and treatment of BBD within the last 5 years. Results from a BBD questionnaire were recorded at each clinic visit as well as the treatment each patient received. A self-administered 5-point Likert scale was used to assess the severity of each patient's BBD symptoms and the results were compared from the patient’s first to final visit. The questionnaire classified BBD symptom severity and frequency into eight subscales. The mean change in each subscale demonstrated the efficacy of each intervention studied. 

Results
Data from 300 patients was reviewed and included in the study. The average patient age was 6.1 years and included 89 males (30%) and 211 females (70%). Stool softeners demonstrated statistically significant improvement in the subscales of regularity of bowel movements, decreased stool accidents, and decreased pain and straining associated with bowel movements. Urotherapy significantly reduced daytime urinary incontinence. Stool softeners and anticholinergics each independently reduced urinary holding and straining. The limitations of the study were the concurrent interventions and subjective nature of the questionnaire data.

Conclusion
In addition to effectively managing constipation symptoms, stool softeners reduced daytime urinary incontinence and improved urinary holding and straining, underscoring the efficacy of stool softeners across multiple domains of BBD and highlighting the interplay between bowel and bladder function. Finally, our study demonstrated that the behavioral intervention of urotherapy was more effective than the medical management of daytime urinary incontinence in pediatric BBD.

Determining Best Treatments for Pediatric Bowel and Bladder Dysfunction

Background 
The exponential growth of biomedical literature, especially in urology, with 1.3M new citations added to MEDLINE in 2023, makes it difficult for clinicians to stay updated and conduct literature reviews and meta-analyses. Artificial intelligence, specifically through a technique called sentiment analysis, can help by facilitating the rapid extraction and summarization of insights from study data. Sentiment analysis uses large language models (such as GPT-4) to classify texts based on sentiment (i.e. tone expressed). This study tests GPT-4 in conducting sentiment analysis of literature about a common urology topic — the utilization of radiotherapy versus prostatectomy as treatment for localized prostate cancer — by comparing GPT-classified sentiments of studies to human-classified sentiments. 

Methods 
A PubMed literature search using MeSH keywords for prostate cancer, prostatectomy, and radiotherapy, yielded 29 relevant studies. Abstracts were reviewed by the research team, with members independently assigning a sentiment of prostatectomy-preferred, radiotherapy-preferred, or neutral. 
GPT-4 was then provided three abstracts that were human-classified as prostatectomy-preferred, radiotherapy-preferred, and neutral. This provided GPT-4 a baseline for sentiment classification. The model subsequently classified the remaining 26 abstracts by sentiment. It was prompted five separate times to ensure consistency. 
GPT-4 sentiment classification results were compared to human-classified sentiments (reference, gold standard answers) through accuracy, precision, recall, and F1 score metrics to gauge model performance. 

Results 
GPT-4 had 80.8% (21/26) accuracy in classifying sentiments compared to manual, human classified sentiments, correctly categorizing 3 radiotherapy-preferred, 7 prostatectomy-preferred, and 11 neutral abstracts. GPT-4 had 76.5% recall, 82.8% precision, and an F1 score of 79.5% (F1 scores range between 0-1, with 75% being an approximate cutoff for “good”). 

Conclusion 
GPT-4 is capable of fairly accurate (80.8%) sentiment analysis, even for specialized domains like urology. Its mistakes were limited to differentiating between neutral and polarized (radiotherapy-preferred or prostatectomy-preferred) sentiments. Mis-categorization of neutrality can be due to lack of specific words deemed neutral, as opposed to positive and negative. Thus, GPT-4 has promise to rapidly parse through large numbers of studies and capture overall sentiment, which may be useful for clinicians. 


Leveraging AI in urologic literature: GPT-4 for sentiment analysis on the utilization of radiotherapy versus prostatectomy in prostate cancer as an upfront treatment

Background: Patients may seek gender-affirming orchiectomy and scrotectomy (GAOS) to alleviate dysphoria related to the scrotum and testes, while allowing for penetrative intercourse, if desired. However, techniques and outcomes of scrotectomy in this specific setting have not been described to date. Here we discuss our initial experience with GAOS as an option for patients who do not necessarily desire other gender-affirming genital surgeries.

Methods: All patients who had undergone GAOS procedures from 2021 to 2022 at our institution were reviewed. The World Professional Association for Transgender Health (WPATH) criteria for surgical treatment were met for all patients preoperatively. In addition, patients were counseled on the irreversibility of the procedure and its implications, including sterility and the impact on future surgical options such as vaginoplasty. Patients were offered a choice of two approaches depending on their surgical goals: excision of a majority of scrotal tissue and primary closure of the perineal wound, or excision of all rugated skin with mons and groin (Y-flap) advancement. Preoperative demographic data, baseline sexual function, intraoperative findings, and postoperative outcomes were collected. Patient-reported outcomes (PROs) derived from the PROMIS ® Sexual Function questionnaires were sent to all patients postoperatively and collected.

Results: Five patients underwent GAOS during the study period. Median age was 29 years and median preoperative duration on hormones was 16 months. All patients reported dysphoria related to the scrotum and testes, and wished to preserve the phallus. Three of the five patients elected for primary closure, while two desired Y-flap perineoplasty. Median operative time was 152 min and median resected scrotal area was 96 cm2; all patients were discharged home the same day. At median follow up of 97 days, all patients reported satisfaction with the surgical outcome. One patient who had Y-flap experienced wound dehiscence requiring operative revision. Three of five patients completed detailed questionnaires regarding resolution of dysphoria and preservation of sexual function. All patients who experienced postoperative complications reported being sexually active with the ability to achieve erection and orgasm within the last thirty days of the survey collection, and no change to their libido. Further patient follow-up is being collected.

Conclusion: GAOS is a well tolerated procedure that can reasonably address dysphoria related to the scrotum and testes. Further and longer-term study of this emerging procedure is warranted.

Scrotectomy as Genital Gender-Affirming Surgery: Novel Surgical Technique and Patient-Reported Outcomes

Objective: 
Varicocele thrombosis has historically been described as a rare phenomenon, and little is known about how COVID-19 and its thrombophilic effects manifest in the male reproductive tract. The purpose of this study is to examine the risk of thrombosis in varicocele patients with or without documented COVID-19 infection.

Design: 
This is a retrospective cohort study composed of patients from a large tertiary care institution. 

Subjects: 
The study cohort was composed of patients with a clinical or radiographic diagnosis of varicocele between 2013-2022. 

Exposure: 
The exposure of interest in this study was documented COVID-19 infection status.

Main Outcome Measures:
Patient data was collected including COVID-19 infection status, age, and BMI via retrospective chart review of the electronic medical record. Pampiniform plexus thrombosis was diagnosed by a staff radiologist and noted within scrotal ultrasonography reports. Scrotal ultrasound reports were analyzed by independent reviewers for diagnosis of varicocele thrombosis. Standard statistical analysis was performed using chi-squared and t-tests in addition to multivariate regression analysis. 

Results:
We identified 110 cases of varicocele thrombosis amongst 1,999 diagnoses of varicocele, representing at least a 5.5% overall incidence rate. There was a strong association between history of COVID-19 infection and varicocele thrombosis (p=<0.001). There was also a significant increase in thrombosis incidence in 2020, consistent with epidemiological data regarding acute COVID-19 infection in unvaccinated reproductive-aged males. 

Conclusion:
Our data suggests that in contrast to prior reports, varicocele thrombosis is a relatively common phenomenon and is present in up to 5.5% of men with varicocele. COVID-19 is a strong risk factor for the development of thrombosis, and more work is necessary to reveal the effect of this phenomenon on long-term testicular function in patients with varicocele. This diagnosis should be considered in men with subacute scrotal pain out of proportion of traditional varicocele discomfort, particularly if a recent COVID-19 infection is suspected or confirmed.

Varicocele Thrombosis is Surprisingly Common and is Strongly Linked to COVID-19 Infection

Introduction:
A commonly used treatment for bipolar disorder is lithium which can cause numerous adverse events due to its low therapeutic index. Though, the literature poorly documents the association between lithium and skin and subcutaneous tissue disorders. To form targeted monitoring and management strategies understanding the distribution and trends of these adverse events in patients using lithium is crucial. The study aims to comprehensively analyze the association between lithium usage and the occurrence of skin and subcutaneous tissue disorders by utilizing data from the FDA Adverse Event Reporting System (FAERS). 

Methods:
A comprehensive search was performed on FAERS for skin and subcutaneous tissue disorder
adverse events due to lithium usage reported from 1973 to 2024. Adverse events were categorized by sex, age group, reporter region, and reporter type. To provide a comprehensive overview of the distribution and trends of these adverse events descriptive statistics and trend analysis were employed. 

Results:
A total of 782 cases of skin and subcutaneous tissue disorders were associated with lithium usage. The distribution by sex revealed a higher number of cases in females (412) compared to males (305), with a smaller proportion of cases in unspecified categories (65). Age group analysis demonstrated that most cases (484) occurred in the 18-64 age group, with a notable concentration in adults. Geographically, the cases were nearly evenly distributed between domestic (384) and foreign (396) reports. Reporter-type analysis indicated that healthcare professionals reported most cases (499), followed by consumers (229) and a small number of unspecified sources (54).

Conclusion:
The findings of this study highlight that skin and subcutaneous tissue disorders associated with lithium usage are more frequently reported in females and adults aged 18-64 years, with healthcare professionals being the primary reporters of these adverse events. These results underscore the importance of targeted monitoring and management strategies for patients on lithium therapy, mainly focusing on demographic groups at higher risk. Further research is warranted to explore the underlying mechanisms and develop preventive measures for these adverse events.

Association Between Lithium Usage and Skin and Subcutaneous Tissue Disorders: Analysis of FAERS Data

Abstract Title: Association of invasive bacterial infections and under-vaccinated status in well-appearing pediatric patients presenting to the emergency department with fever. 

Background: Fever accounts for up to 40% of visits to the pediatric emergency department (ED). While fever can be caused by viral or bacterial infections, those developing invasive bacterial infections (IBI) are at increased risk for life-threatening complications. Thus, evaluating a patient’s risk factors—including assessment of vaccination status—for developing IBI is an essential step. For example, invasive illness from Haemophilus influenzae (Hib) was the leading cause of childhood bacterial meningitis prior to the advent of the vaccine, with around 200,000 cases occurring yearly. Over the decade following the vaccine introduction, the prevalence of invasive disease from bacteria, such as Hib, had been almost eradicated. This study aims to determine the association between vaccination status and IBI in well-appearing pediatric patients presenting to the ED with a chief complaint of fever. 

Methods: This is a retrospective cohort study conducted between 2019-2023, at a single, urban tertiary care pediatric emergency department. Patients were included if their age was between 61 days and 18 years old presenting with a chief complaint of fever. Patients were excluded if they received antibiotics within 3 days or underwent surgery within 2 weeks of presentation. IBI was defined as bacteremia, bacterial meningitis, septic arthritis, osteomyelitis, pneumonia, deep neck space infection, mastoiditis, or orbital cellulitis. Prevalence of IBI was described and compared by presence of age group, chronic medical condition, gender, and vaccination status. Logistic regression was used to analyze the association between IBIs and vaccination status. 

Results: 2,623 patients were screened, and 1249 patients were included. Amongst patients who were included, the prevalence of IBI in the study was 11.9%. Of the 80 under-immunized patients, nine (11.25%) developed an IBI. One hundred and forty (12%) of the 1,169 fully immunized patients developed an IBI. The odds ratio is 1.07 (95%CI 0.52-2.20). 

Conclusion: In conclusion, the preliminary data does not show a significant difference in risk of IBI in patients who are under-immunized versus those who are fully immunized. Thus, clinicians should exercise caution using vaccination status alone to determine risk of IBI in well-appearing febrile pediatric patients presenting to the emergency department. However, low rates of IBI and under-vaccinated status as well as herd immunity in our population may indicate sample bias. Our future research aims to increase the sample size to further investigate this concept.

Association of Invasive Bacterial Infections and Under-Vaccinated Status in Well-Appearing Pediatric Patients

Background
Enterococcus faecalis (E. faecalis) is a well-known bacterium that colonizes the human gastrointestinal (GI) tract. It is most recognized for causing antibiotic-resistant bacteremia, pneumonia, intra-abdominal infections, and endocarditis. Overgrowth in the gut and translocation to the bloodstream through the intestinal epithelium is the widely accepted mechanism for developing E. faecalis bacteremia, although the mechanisms of invasion, survival, and virulence continue to be investigated.

Case Presentation
A 37-year-old man with a past medical history of acute monocytic leukemia (AML),
hypertension, and type II diabetes mellitus presented to the hospital with worsening chills, diffuse muscle aches, increased drainage from mouth sores, subjective fevers, and increasing fatigue six days after completing a 7+3 chemotherapy regimen with daunorubicin/cytarabine. The patient had gingivostomatitis predating his official AML diagnosis. Febrile neutropenia was suspected due to the patient's symptoms and pancytopenia with prolonged and profound neutropenia. Blood cultures from admission grew E. faecalis repeat blood cultures were negative, and no aerobic bacteria were visualized. Respiratory cultures, Legionella antigen, rapid influenza A and B, and COVID-19 PCR tests were all negative. The patient was initially
empirically treated with cefepime, then switched to piperacillin/tazobactam. When
susceptibilities from the initial blood culture showed that E. faecalis was pan-sensitive, the patient was discharged on an oral regimen of amoxicillin for 14 days. Repeat blood cultures were negative within 48 hours of discharge. A transthoracic echocardiogram showed no evidence of vegetations, and there was low suspicion for infective endocarditis.

Discussion
There have been few documented cases of E. faecalis associated with underlying colorectal malignancies and the development of infective endocarditis. However, there are no documented cases where E. faecalis was identified in the bloodstream and treated before complications (e.g., infective endocarditis) developed. The scarcely documented cases of pan-sensitive E. faecalis make this case unique. The translocation of E. faecalis from the GI tract to the bloodstream is possibly more likely during induction chemotherapy with daunorubicin and cytarabine, which damage the gastrointestinal epithelium and cause painful oral mucositis and diarrhea. In combination with the neutropenia induced by this treatment, it is very likely that this patient’s AML chemotherapy regimen led to his E. faecalis infection. The feasibility of gut-protective measures during intensive chemotherapy induction to reduce translocation of gastrointestinal infections is an area needing further investigation. The patient’s rapid recovery from this infection and its pan-susceptibility is unusual and resulted in an excellent outcome.

Blood Stream Invasion: Enterococcus Faecalis Takes a Wild Gut Trip in a Neutropenic
Environment

Introduction: 
Aortic calcification is a significant pathological process associated with various cardiovascular diseases. While the sympathetic nervous system's influence on bone remodeling via β2-adrenergic receptors (β2-ARs) is well-established, its impact on vascular calcification remains relatively unexplored. Previous research suggests β2-ARs play a critical role in regulating valve calcification but have yet to be studied in aortic calcification. Thus, we aim to characterize the expression patterns of both β1-ARs and β2-ARs to elucidate the downstream signaling mediators in human aortic tissue and determine the effects of selective β2-AR agonists on aortic smooth muscle cell phenotype and function. 

Methods: 
Calcified and non-calcified abdominal aortic wall segments from 6 patients with abdominal aortic aneurysm (AAA) were collected during elective open aortic repair at Northwestern Memorial Hospital (Chicago, IL). Samples were fixed, decalcified, and embedded in paraffin. Sections of 5 μm thickness were used for immunostaining of alpha-smooth muscle actin, β1-AR, and β2-AR. Human aortic smooth muscle cells (HAoSMC) were cultured in osteogenic media and treated with one of the following β-AR agonists (1 - 100 μmol/L): salmeterol, isoproterenol, norepinephrine, or an antagonist (0.3 – 30 μmol/L): ICI-118,551 or CGP-20712A. Cells were fixed for immunocytochemical staining of osteogenic markers (osteocalcin, osteopontin, and Runx2) and β-ARs. HAoSMCs were treated with salmeterol (1 μmol/L, β2-AR agonist) in the presence of either growth media or osteogenic media for up to 14 days. Metabolic and alkaline phosphatase (ALP) activity were measured with resazurin (n=8) and ALP assays (n=3), respectively, on days 7 and 14. 

Results: 
Samples of abdominal aortic wall segments show differential expression of β2-adrenergic receptors between patients with abdominal aortic aneurysms. Culturing human aortic smooth muscle cells (hAoSMCs) in osteogenic (OST) media for 21 days successfully induced an osteogenic phenotype as evidenced by increased alkaline phosphatase (ALP) activity over time. Delivery of β2 agonist salmeterol to human aortic smooth muscle cells cultured in both growth media and osteogenic media showed no significant difference in metabolic activities after 21 days. Osteogenic cells treated with salmeterol demonstrate less alkaline phosphatase activity at day 21. Osteogenic differentiation of human aortic smooth muscle cells downregulates β2-adrenergic receptor expression. Stimulation of osteogenic smooth muscle cells with a β2 agonist, salmeterol, further downregulates receptor expression. Osteopontin expression decreased in osteogenic human aortic smooth muscle cells treated with salmeterol.

Conclusions: 
We observed a differential expression of β2-ARs across sections of human calcified aortas, suggesting a role of these receptors in areas subjected to different calcific stresses. In vitro experiments also demonstrated an impact on the calcification process. Further in vitro experiments with HAoSMCs treated with β-AR agonists and antagonists will help to elucidate the influence of β-adrenergic signaling on vascular calcification pathways. 


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