Citations

Increases in local angiotensin II (ANGII) signaling have been implicated in a number of diseases, including atherosclerosis, hypertension, and aneurysm. Endothelial dysfunction is strongly associated with increased ANGII signaling and the pathogenesis of these diseases. While increases in Runx2 have been implicated in cardiovascular calcification and vascular stiffening in diabetes, little is known regarding the role of Runx2 in the regulation of endothelial function. In the present study, we hypothesized that Runx2 is a key mediator of impaired vascular function and increased vascular wall stiffness provoked by ANGII. Wild-type mice (WT) and littermates that were deficient in one copy of Runx2 (HET) were implanted with osmotic minipumps containing saline or a pressor dose of ANGII (1000 ng/kg/min). Blood pressure was measured non-invasively using tail-cuff methods (CODA) and mice were sacrificed following 14 days of treatment. Changes in endothelium-dependent relaxation (relaxation to acetylcholine, R ACH ) and passive pressure-diameter relationships were measured in vitro (Living Systems arteriograph). In WT mice, treatment with ANGII for 14 days resulted in profound impairment in R ACH (85 ± 4% vs 47 ± 14% p<0.05) and slight reductions in vascular diameter at high distending pressures (~10% reduction in maximal diameter). While Runx2 deficiency did not alter R ACH or vascular distensibility/compliance in saline-treated mice, loss of one copy of Runx2 in ANGII-treated mice dramatically improved R ACH compared to their wild-type, ANGII-treated littermates. Interestingly, impairments in the pressure-diameter relationship with ANGII infusion were not affected by loss of one copy of Runx2. Critically, reductions in Runx2 did not significantly alter the pressor response to ANGII. In conclusion, our data suggest that Runx2 may play an integral role in the pathogenesis of ANGII-induced endothelial dysfunction, with these changes occurring independently of alterations in extracellular matrix remodeling and vascular stiffness. These data suggest that Runx2 may play a key, context-dependent role mediating vascular responses to ANGII.

Thoracic aortic aneurysm (TAA) is a degenerative condition of the aorta characterized by aortic dilatation secondary to tissue inflammation and matrix remodeling, which ultimately progresses to aortic dissection and rupture. While recent work suggests that in vitro exposure to pro-inflammatory stimuli promotes DNMT3b-dependent DNA methylation in skeletal muscle, epigenetic mechanisms regulating gene expression in TAA remain largely uncharacterized. We therefore tested the hypothesis that expression of tumor necrosis factor α (TNF-α) is increased in human TAA tissue and that such increases are associated with upregulation of DNA methyltransferase (DNMT) levels. Normal aortic samples (n=13) were acquired from non-aneurysmal hearts/aorta not suitable for cardiac transplant, whereas TAA samples from patients with tricuspid aortic valves (n=29) were acquired from patients undergoing elective surgery for aortic aneurysm. We measured relative expression of TNF-α, DNMT1, DNMT3a and DNMT3b mRNA levels in normal and TAA tissue by qRT-PCR, and DNMT3b protein levels by western blot. Marked increases in expression of the inflammatory marker TNF-α were noted in TAA tissue (12.3± 2.34), and were associated with significantly increased DNMT1 (1.4±0.13), DNMT3a (1.44±0.09) and DNMT3b (1.94±0.27) mRNA levels (p<0.05 for all). DNMT3b protein levels were also dramatically increased in human TAA samples (2.99±0.58-fold, n = 10/group, p<0.05) when compared to normal aortic tissue. Collectively, these data suggest a novel potential role for DNA methyltransferases as major regulators of altered gene expression in TAA, and support our working hypothesis that inflammation-induced increases in DNMT3b levels may be critical repressors of protective gene expression in TAA.

Mitochondrial antioxidant genes are major regulators of oxidative stress in the vasculature. Previous data has shown that reducing antioxidant capacity accelerates atherosclerotic plaque size, however, it is unclear whether reducing mitochondrial antioxidant capacity alters plaque composition. Therefore, we tested the hypothesis that reductions of mitochondrial antioxidant capacity will increase osteogenic markers and intimal plaque calcification in aorta from hypercholesterolemic mice. We used Ldlr -/- ApoB 100/100 (LA) mice that were either wild-type (LA-MnSOD +/+ ) or heterozygous (LA-MnSOD +/- ) for manganese superoxide dismutase that were fed a Western Diet (TD88137) for 3 or 6 months. Changes in changes gene expression were assessed in aortic arch using qRT-PCR, plaque calcium levels were examined using histological evaluation from aortic cryosections, and endothelial function was measured using isolated organ bath chambers ( ex vivo ). While expression of SP7 (an osteogenic transcription factor) was unchanged between LA-MnSOD +/+ (1.0±0.6) and LA-MnSOD +/- (0.95±0.3) at 3 months, SP7 levels were dramatically increased in LA-MnSOD +/+ (8.0±2.8) at 6 months and increased further in LA-MnSOD +/+ mice (17.5±5.9; p<0.05). Alizarin Red staining for calcium showed increased calcium burden in plaques of 6 month LA-MnSOD +/- compared to LA-MnSOD +/+ (3636±669 pixels versus 649±206 pixels; p<0.05). Vasomotor function in aorta was unchanged between LA-MnSOD +/+ and LA-MnSOD +/- mice, suggesting that the transcriptional and functional changes related to calcification in LA-MnSOD +/- mice occur independently of nitric oxide signaling. In conclusion, our data strongly support our working hypothesis that reductions in mitochondrial antioxidant capacity significantly accelerate osteogenic calcification in aorta from hypercholesterolemic mice, and suggest that targeting mitochondrial oxidative stress may be a viable therapeutic strategy to slow vascular calcification.

Introduction While progression of calcific aortic valve stenosis (CAVS) is known to be strongly associated with activation of pro-inflammatory and pro-osteogenic pathways, effective treatments for CAVS have remained elusive. TNF-α is a pleiotropic inflammatory cytokine and is upregulated in patients with CAVS. Hence, we hypothesized that TNF-α inhibition, using the neutralizing antibody infliximab, can slow the progression of CAVS in vivo . Methods We used Ldlr -/- Apob 100/100 mice, which develop robust CAVS in an age-dependent manner, as a model. Mice were fed a Western-type diet for 6 months, then randomized into one of two groups: 1) twice-weekly infliximab injections (10 mg/kg intraperitoneally; n=10), and 2) saline injections (n=9). Aortic valve function was evaluated using echocardiography, and gene expression in aortic valves was measured using quantitative real-time PCR. Data are presented as mean ± SEM. Results During the 3 months preceding treatment initiation, aortic valve cusp separation distance decreased progressively in both groups (reduction by 0.082±0.011 mm in control group, 0.095±0.016 mm in infliximab group; p =0.27). Three months after initiation of treatment, cusp separation continued to decrease in control mice (by 0.108±0.021 mm), while progression of valve dysfunction was slower by 42% in the infliximab group (reduction of 0.063±0.009 mm; p =0.03 vs control). Expression of the pro-inflammatory genes Vcam1 and Nos2 was significantly reduced in valves of the infliximab group (by 37% and 60%, respectively). Moreover, endothelium-dependent vasodilation was improved by infliximab (improvement in maximum relaxation to acetylcholine in infliximab group 18±6% compared to control). Conclusions This study is the first to report that in vivo inhibition of TNF-α slows progression of CAVS in mice with established valve disease. This beneficial effect was accompanied by reduction in pro-inflammatory gene expression and improvement of systemic endothelial function, suggesting infliximab may exert its effects in part by increasing nitric oxide bioavailability. The findings warrant further investigation into the role of TNF-α in initiation and progression of CAVS, as well as its potential as a therapeutic target in human CAVS.

Increases in mitochondrial reactive oxygen species (ROS) can contribute to endothelial dysfunction in conduit arteries by reducing nitric oxide bioavailability. SIRT3 is a protein deacetylase localized to mitochondria and regulates activity of numerous electron transport chain enzymes, and can be inactivated by increases in mitochondrial ROS. Whether reductions in SIRT3 are an independent contributor to endothelial dysfunction, however, are unknown. Therefore, we hypothesized that SIRT3 null mice (SIRT3 -/- ) will have impaired relaxation to acetylcholine (MR Ach ) compared to wild-type littermates (SIRT3 +/+ ), and that this difference will be exacerbated by Western diet feeding for 3 months (TD88137, 42% of kcal from fat, 0.2% cholesterol). Endothelial function was measured using isolated organ bath chambers, and endothelium-dependent (MR Ach ) and endothelium-independent (sodium nitroprusside, MR SNP ) relaxation were measured. In contrast to our hypothesis, MR ACH was virtually identical between SIRT3 +/+ and SIRT3 -/- fed a standard chow diet (MR ACH = 68±2% and 69±3%; respectively), and MR SNP also remained unchanged between the two genotypes. In groups fed a Western diet, however, endothelium-dependent relaxation was significantly impaired in SIRT3 -/- mice compared to SIRT3 +/+ littermates (MR ACH = 49±4% and 58±3%, respectively; p<0.05). MR SNP was not altered by either Western diet feeding or SIRT3 deficiency. Interestingly, impairments in MR Ach in SIRT3 -/- mice fed a Western diet were not rescued with acute incubation of apocynin (an NAD(P)H oxidase inhibitor) or mito-tempol (a mitochondria-targeted antioxidant), suggesting that chronic antioxidant and/or acetyltransferase inhibition may be required to improve vasomotor function in this model. Collectively, our data suggest that SIRT3 plays a major role in regulating endothelial function with dietary stress, and may be a useful therapeutic target to reduce cardiovascular risk.

Abstract Background Orthopaedic surgery has the lowest proportion of women surgeons in practice of any specialty in the United States. Preliminary studies suggest that patients who are treated by physicians of the same race, ethnicity, cultural background, or gender feel more comfortable with their care and may have better outcomes. Therefore, understanding the discrepancies in the diversity of the orthopaedic surgeon workforce is crucial to addressing system-wide healthcare inequities. Questions/purposes (1) Does a difference exist in gender representation among practicing orthopaedic surgeons across geographic distributions and years in practice? (2) Does a difference exist in gender representation among practicing orthopaedic surgeons with regard to rural-urban setting, group practice size, and years in practice? Methods Orthopaedic surgeons serving Medicare patients in 2017 were identified in the Medicare Physician and Other Supplier Public Use File and Physician Compare national databases. This dataset encompasses more than 64% of practicing orthopaedic surgeons, providing a low proportion of missing data compared with other survey techniques. Group practice size, location, and Rural-urban Commuting Area scores were compared across physician gender and years in practice. Linear and logistic regressions modeled gender and outcomes relationships adjusted by years in practice. Least-square means estimates for outcomes were calculated by gender at the median years in practice (19 years) via regression models. Results According to the combined Medicare databases used, 5% (1019 of 19,221) of orthopaedic surgeons serving Medicare patients were women; this proportion increased with decreasing years in practice (R2 0.97; p < 0.001). Compared by region, the West region demonstrated the highest proportion of women orthopaedic surgeons overall (7% [259 of 3811]). The Midwest and South regions were below the national mean for proportions of women orthopaedic surgeons, both overall (5% [305 of 6666] and 5% [209 of 4146], respectively) and in the first 5 years of practice (9% [54 of 574] and 9% [74 of 817], respectively). Women worked in larger group practices than men (median [interquartile range] 118 physicians [20 to 636] versus median 56 [12 to 338]; p < 0.001, respectively). Both genders were more likely to practice in an urban setting, and when controlling for years in practice, there was no difference between men and women orthopaedic surgeons practicing in rural or urban settings (respectively, R2 = 0.0004 and 0.07; p = 0.89 and 0.09). Conclusion Among orthopaedic surgeons, there is only one woman for every 20 men caring for Medicare patients in the United States. Although gender representation is increasing longitudinally for women, it trails behind other surgical subspecialties substantially. Longitudinal mentoring programs, among other evidenced initiatives, should focus on the more pronounced underrepresentation identified in Midwestern/Southern regions and smaller group practices. Gender-based equity, inclusion, and diversity efforts should focus on recruitment strategies, and further research is needed to study how inclusion and diversity efforts among orthopaedic surgeons improves patient-centered care. Level of Evidence Level III, therapeutic study.

Background Combining immunotherapy and antiangiogenic agents is a promising treatment strategy in endometrial cancer. To date, no biomarkers for response have been identified and data on post-immunotherapy progression are lacking. We explored the combination of a checkpoint inhibitor (nivolumab) and an antiangiogenic agent (cabozantinib) in immunotherapy-naïve endometrial cancer and in patients whose disease progressed on previous immunotherapy with baseline biopsy for immune profiling. Patients and methods In this phase II trial (ClinicalTrials.gov NCT03367741, registered December 11, 2017), women with recurrent endometrial cancer were randomized 2:1 to nivolumab with cabozantinib (Arm A) or nivolumab alone (Arm B). The primary endpoint was Response Evaluation Criteria in Solid Tumors-defined progression-free survival (PFS). Patients with carcinosarcoma or prior immune checkpoint inhibitor received combination treatment (Arm C). Baseline biopsy and serial peripheral blood mononuclear cell (PBMC) samples were analyzed and associations between patient outcome and immune data from cytometry by time of flight (CyTOF) and PBMCs were explored. Results Median PFS was 5.3 (90% CI 3.5 to 9.2) months in Arm A (n=36) and 1.9 (90% CI 1.6 to 3.4) months in Arm B (n=18) (HR=0.59, 90% CI 0.35 to 0.98; log-rank p=0.09, meeting the prespecified statistical significance criteria). The most common treatment-related adverse events in Arm A were diarrhea (50%) and elevated liver enzymes (aspartate aminotransferase 47%, alanine aminotransferase 42%). In-depth baseline CyTOF analysis across treatment arms (n=40) identified 35 immune-cell subsets. Among immunotherapy-pretreated patients in Arm C, non-progressors had significantly higher proportions of activated tissue-resident (CD103+CD69+) ɣδ T cells than progressors (adjusted p=0.009). Conclusions Adding cabozantinib to nivolumab significantly improved outcomes in heavily pretreated endometrial cancer. A subgroup of immunotherapy-pretreated patients identified by baseline immune profile and potentially benefiting from combination with antiangiogenics requires further investigation.

Abstract Context Enhanced Recovery After Surgery (ERAS) is a multimodal protocol aimed to improve quality of postoperative recovery, minimize complications, and optimize overall self-regulation. Preoperative gabapentin decreases postoperative pain but can be associated with prolonged postoperative somnolence and respiratory depression risk. Although it is known that gabapentin affects the postoperative course, it is unclear if the timing of preoperative administration affects this finding. Objectives This study aims to assess the optimal preoperative timing for gabapentin administration in patients undergoing gynecologic surgery to minimize postoperative somnolence risk. Methods A retrospective cohort study evaluated patients who underwent major gynecologic surgery and received preoperative gabapentin. Patients were grouped based on timing from gabapentin administration to surgical incision (<4 h group vs. ≥4 h group). Preoperative, intraoperative, and postoperative data were abstracted and compared. Univariate associations between the timing of gabapentin administration and the patient and surgical characteristics and outcomes were tested utilizing two-sample equal-variance t-tests, linear model ANOVA, or Fisher’s exact tests. Associations between the timing of gabapentin administration and the time until the Richmond Agitation Sedation Scale (RASS) score of 0 were modeled utilizing linear regression, adjusted for age, initial postoperative anesthesia care unit (PACU), RASS score, and postoperative narcotics. Results Each group contained 127 patients. Demographics were similar except for age (<4 h group mean=44.2 years; ≥4 h group mean=40.5 years; p=0.021), chronic pain (<4 h group=17.6%; ≥4 h group=43.3%; p<0.001), and surgical indication (<4 h group=pelvic pain [29.1%]; ≥4 h group=pelvic pain [51.2%]; p=0.007). The <4 h group had a similar postoperative narcotic administration (<4 h group mean morphine milligram equivalents [MME]=3.667; ≥4 h group mean MME=4.833; p=0.185). The minutes from surgical closure until the patient received a RASS score of 0 and initial PACU pain score (Visual Analogue Scale [VAS]) were similar. The initial PACU oxygen administration volume, hours from surgical closure until the patient transitioned to room air, and initial PACU respiratory rate were similar. The PACU duration, admission secondary to somnolence, and initial PACU Glasgow Coma Scale (GCS) score showed no difference. Postoperative nausea/vomiting was decreased in the ≥4 h group (<4 h group=24.4%; ≥4 h group=13.4%; p-value=0.036), and urinary retention (<4 h group=14.2%; ≥4 h group=5.5%; p-value=0.033) was decreased in the ≥4 h group. Conclusions The timing of gabapentin administration less than or more than 4 h preoperatively in patients ≥18 years does not significantly affect postoperative somnolence or respiratory depression. Further, it does not have a significant effect on GCS scores or VAS scores.