Luteolin-Mediated Inhibition of Hepatic Stellate Cell Activation via Suppression of the STAT3 Pathway.

Hepatic stellate cell (HSC) activation is responsible for hepatic fibrogenesis and is associated with an overexpression of transcription 3 (STAT3). Luteolin, a common dietary flavonoid with potent anti-inflammatory properties, has previously demonstrated antifibrogenic properties in HSCs but the mechanism has not been fully elucidated. Activated human and rat hepatic stellate cell lines LX-2 and HSC-T6 were used to study the effects of luteolin on HSCs. Cellular proteins were determined by western blot and immunofluorescence. Cell proliferation was assessed with Alamar Blue assay. Luteolin significantly decreased LX-2 and HSC-T6 cell viability in a time-and-dose-dependent manner, as well as decreased HSC end-products α-smooth muscle actin (α-SMA), collagen I, and fibronectin. Luteolin decreased levels of total and phosphorylated STAT3, suppressed STAT3 nuclear translocation and transcriptional activity, and attenuated expression of STAT3-regulated proteins c-myc and cyclin D1. STAT3 specific inhibitors stattic and SH-4-54 demonstrated similar effects on HSC viability and α-SMA production. In LX-2 and HSC-T6 cells, luteolin demonstrates a potent ability to inhibit hepatic fibrogenesis via suppression of the STAT3 pathway. These results further elucidate the mechanism of luteolin as well as the effect of the STAT3 pathway on HSC activation.

Generation of the First Transgenic Line of the Iconic Coral Reef Fish Amphiprion ocellaris.

The common clownfish, Amphiprion ocellaris, is an iconic coral reef fish, ubiquitous in the marine aquarium hobby and useful for studying a variety of biological processes (e.g., mutual symbiosis, ultraviolet vision, and protandrous sex change). Recently, CRISPR/Cas9 methods were developed for knocking out specific genes for mechanistic studies. Here, we expand the genetic toolkit for A. ocellaris by creating the first transgenic line using the Tol2 transposon system. Fertilized eggs were co-injected with Tol2 transposase mRNA and a plasmid encoding an elongation factor-1α (Ef1α): green fluorescent protein (GFP) cassette at various concentrations, needle tip dimensions, and timepoints post-fertilization. We compared various injection parameters and sterilization methods to maximize the survival of injected eggs. F0s (n = 10) that were genotyped GFP + were then raised to 6 months of age and crossed with wild-type (WT) females to confirm germline transmission. F1 offspring were also raised and crossed in the same manner. The highly efficient Tol2 transposon system resulted in a 37% rate of transgenesis for surviving eggs amounting to a 2.7% yield of all injected eggs surviving and being GFP + (n = 160). Of these, 10 were raised to adulthood, 8 spawned, and 5/8 (62.5%) produced GFP + offspring. Further, two F1s crossed with WT females produced 54.2% and 44.6% GFP + offspring respectively, confirming the creation of a stable line. This is, to our knowledge, the first generation of a transgenic line in any coral reef fish. The ability to express transgenes of interest in the iconic anemonefish opens the door to a new era of exploration into their fascinating biology.

Outcomes of Biventricular and Single Ventricle Heterotaxy Patients: A Single Center Five-Decade Experience.

BACKGROUND: Surgical palliation of patients with heterotaxy syndrome has proven challenging. Long-term outcomes have historically been poor. Factors contributing to these outcomes are not completely understood. METHODS: The institutional databases were queried for patients with heterotaxy syndrome from 1973 to 2021. Comparisons were made between patients managed with single ventricle physiology and biventricular physiology. RESULTS: Heterotaxy syndrome was identified in 230 patients (polysplenia, 47%; asplenia, 53%). In all, 199 patients had single ventricle physiology; 180 (78%) had undergone Fontan palliation. Thirty-one patients had biventricular physiology, including 20 (9%) with surgical intervention and 11 (5%) without surgical intervention. Median age at Fontan was 7.5 years (interquartile range, 8.8). Median follow-up was 20 years (interquartile range, 21). Kaplan-Meier analysis showed decreased survival with single ventricle physiology (53% ± 4%, vs biventricular 93% ± 5% at 30 years; P = .001), as well as asplenia compared with polysplenia (49% ± 5% vs 68% ± 5% at 30 years; P < .001). Polysplenia patients with biventricular physiology demonstrated the best survival (100% alive, vs 53% ± 25% of asplenia biventricular at 30 years; P < .001). Overall, 8 patients (3.5%) underwent cardiac transplantation at a median age of 17 years. On multivariable analysis, risk factors associated with mortality included single ventricle physiology (odds ratio [OR] 7.2; 95% CI, 2.4-21.7), no prior Glenn (OR 3.6; 95% CI, 1.9-6.7), need for permanent pacemaker (OR 2.3; 95% CI, 1.2-4.6), and asplenia (OR 2.7; 95% CI, 1.5-5.0). CONCLUSIONS: Overall, patients with asplenia demonstrated decreased survival compared with patients with polysplenia; and single ventricle physiology had decreased survival compared with biventricular. Patients with biventricular physiology and polysplenia had the best survival.

Partial Anomalous Pulmonary Venous Connection With Intact Atrial Septum: Early and Midterm Outcomes.

BACKGROUND: Partial anomalous pulmonary venous return with intact atrial septum warrants greater understanding and evaluation in the literature. METHODS: From January 1993 to December 2018, 293 patients with partial anomalous pulmonary venous return underwent surgical repair. Of these, 45 patients (15.3%) had an intact atrial septum. The median age was 36 years (interquartile range, 24-48). Direct reimplantation was used in 17 patients (38%), intracardiac baffling in 15 (33%), and caval division (Warden) technique in 13 (29%). Descriptive statistics were used to assess the data, and Kaplan-Meier analysis was used to assess survival. RESULTS: Anomalous veins were right-sided in 27 patients (60%), left-sided in 16 patients (36%), and bilateral in 2 patients (4%). The insertion sites were the superior vena cava in 23 patients (51%), innominate vein in 12 (27%), inferior vena cava in 6 (13%), coronary sinus in 2 (4%), right atrium in 1 patient (2%), and unknown in 1 patient (2%). Scimitar syndrome was noted in 8 patients (18%). There was no postoperative mortality or residual defects. Postoperative echocardiography excluded any obstruction of pulmonary or systemic veins. Postoperative complications included atrial fibrillation in 9 patients (20%) and pneumothorax requiring chest tube in 5 patients (11%). Survival at 1, 5, and 10 years was 100%, 95%, and 95%, respectively. Two patients underwent pulmonary vein dilation, one at 3 years and the other at 7 years. CONCLUSIONS: Surgical repair of partial anomalous pulmonary venous connection with intact atrial septum can be performed with excellent early and midterm outcomes. The overall incidence of midterm systemic or pulmonary vein stenosis is low.

Fontan Palliation in Patients with Heterotaxy Syndrome: A Five Decade Experience.

BACKGROUND: Patients with heterotaxy syndromes (right and left atrial isomerism) are at high risk of poor outcomes after single-ventricle palliation. However, the long-term outcomes and specific parameters associated with poor outcomes are incompletely understood. METHODS: A retrospective review was performed of all patients with atrial isomerism who had a Fontan at our institution from 1973 to 2020. Standard demographic, as well as pre-, peri-, and postoperative parameters were collected. Features and outcomes of patients with polysplenia were compared to asplenia. Outcomes were analyzed for effect during 4 eras: (1: 1973-1984; n = 27), (2: 1985-1994; n = 93), (3: 1995-2004; n = 28), and (4: 2005-2020; n = 10). RESULTS: Of the 1176 patients who had a Fontan operation, 158 (14%) had a heterotaxy syndrome. The median age at the time of Fontan was 8 (9) years. Early mortality was 20% and was greater in patients with asplenia compared to polysplenia (27% vs. 12%, p = .019). But it substantially improved over time (61% in era 1 vs. 7%-10% in the more recent eras (p < .001)), as did transplant-free survival (22% at 10 years in era 1 vs. 88% in era 4, p < .001). Transplant-free survival was significantly lower in patients with asplenia versus those with polysplenia (p = .014), and patients with heterotaxy had lower survival than nonheterotaxy (p = .01). This was largely due to the asplenia group (p < .001) (hazard ratio = 3.05, p = .007). CONCLUSIONS: After Fontan operation, patients with heterotaxy, particularly asplenia, continue to demonstrate worse transplant-free survival than nonheterotaxy patients. Early mortality and long-term transplant-free survival have improved in more recent eras.

Early and Mid-Term Outcomes of Coronary Artery Bypass Grafting in Adults With Congenital Heart Disease.

Atherosclerotic coronary artery disease (CAD) is well-studied in acquired cardiac diseases; however, little data exist regarding outcomes of adult congenital heart disease (ACHD) with CAD. This study examined patients who underwent coronary artery bypass grafting (CABG) during repair of ACHD. This was a retrospective study of patients who underwent CABG for CAD concomitant with ACHD repair 1972-2021. Demographic information, ACHD diagnosis, surgical history, operative details, and outcomes were analyzed. Data are presented as median (interquartile range [IQR]). 157 patients were identified with a median age of 63 (IQR 17) years. Left anterior descending (LAD) was the predominant diseased artery (109 patients [69%]); of those 83 (76%) were treated with mammary artery. 90 (57.3%) patients had 1 bypass, 42 (26.7%) 2, 19 (12%) 3, and 6 (3.8%) had 4. There has been no early mortality since 1988. There was no long-term survival difference between the patients with LAD disease treated with mammary compared to vein (P = 0.68), but early mortality was higher in those treated with vein (10.3% vs 0%, P = 0.018). Late recurrent angina was found in 18 patients (12%) and recurrent CAD found in 17 patients (11%), with 16 patients (10%) requiring CAD reintervention. At most recent follow-up (7.2 [IQR 11.4] years), 101 (64.3%) patients were deceased at 10 (IQR 13.1) years after surgery. Surgical revascularization for CAD may be necessary during the treatment of ACHD, most commonly for LAD disease. Early mortality was low in recent decades. Continued surveillance for recurrent CAD is required.

Early and Late Outcomes of the Warden and Modified Warden Procedure.

BACKGROUND: Operative repair of partial anomalous pulmonary venous connection (PAPVC) remains challenging due to risks of sinus node dysfunction, baffle obstruction, and superior vena cava (SVC) obstruction. METHODS: Traditional or modified Warden procedures were performed in 75 of 318 consecutive patients (24%) with PAPVC repaired surgically at our institution during 1993 to 2021. Clinical characteristics, echocardiography data, operative details, and early and late outcomes were collected. Cumulative incidence of reintervention and Kaplan-Meier survival analysis are reported. RESULTS: Median age was 39 years (interquartile range, 21-57 years). Fifty-nine (79%) had normal sinus rhythm preoperatively. Seventeen (23%) had intact atrial septa. Traditional and modified Warden procedures were performed in 15 (20%) and 60 (80%), respectively. Frequent concomitant procedures included 15 (20%) tricuspid valve repairs and 12 (16%) atrial fibrillation procedures. There were no early deaths. Postoperative complications included atrial fibrillation in 17 (23%), sinus node dysfunction in 15 (20%), pneumothorax in 3 (4%), pleural effusion in 2 (3%), and pacemaker implantation in 1 (1%). At hospital discharge, sinus node dysfunction persisted in 8 (11%). Over a median follow-up of 6 years (interquartile range, 4-10 years), baffle obstruction developed in 1 patient and SVC obstruction developed in 7. None required reoperation and 6 were treated with SVC stents. At 1, 5, and 10 years, the cumulative incidence of reintervention was 5%, 7%, and 14%, and survival was 99%, 94%, and 94%, respectively. CONCLUSIONS: Traditional and modified Warden procedures can be performed with satisfactory early and late survival. Persistent sinus node dysfunction and need for permanent pacing are low. Late SVC obstruction is uncommon and can often be managed nonoperatively.

Systemic Semilunar Valve Repair/Replacement With Fontan Circulation: The Mayo Clinic Experience.

Background: There is a paucity of literature regarding systemic semilunar valve (SSLV) dysfunction in patients with Fontan circulation. We sought to describe our center's 47-year experience with systemic semilunar valve replacement or repair (SSLVR) in patients with Fontan circulation. Methods: The Mayo Clinic Fontan Database is a comprehensive institutional database that stores clinical information of 1176 patients from 1973 to 2021. It was reviewed to identify patients who had a SSLV intervention at the time of or after Fontan. A cohort of 15 patients was identified and a retrospective review of their records was performed. Results: Fourteen patients had SSLV replacement (all mechanical) and one had a repair. SSLVR occurred up to 29 years following the Fontan (mean 11.3 ± 9 years, median 14 years). Thirteen of 14 with SSLVR were performed after Fontan and one was done at the time of initial Fontan. This was an older cohort and mean age at the time of Fontan was 8.7 ± 9.4 years (median 4 years). Indication for the operation was > moderate SSLV regurgitation in all patients. Six patients had decreased ventricular function (EF < 50%) prior to SSLVR and 8 had reduced function after SSLVR. Conclusion: Fortunately, the need for SSLV intervention after Fontan was rare, as evidenced by our small cohort extracted from a large single-institution database spanning a 47-year time period. Reduced preoperative and postoperative ejection fraction was common but did not seem to impact the outcome. Optimal timing for SSLV intervention after Fontan remains unclear.

Early and Late Outcomes of Cardiovascular Surgery in Patients With Ehlers-Danlos Syndrome.

BACKGROUND: Cardiovascular surgical outcomes reports are few for vascular type IV of Ehlers- Danlos Syndrome (vEDS) compared to non-vascular types I-III (nEDS). METHODS: To define cardiovascular surgical outcomes among adult patients (≥18 years) with EDS types, a review of our institution's in-house STS Adult Cardiac Surgery Database-compliant software and electronic medical records from Mayo Clinic (1993-2019) was performed. Outcomes were compared for vEDS patients and nEDS patients. Demographics, baseline characteristics, operative, in-hospital complications and follow-up vital status were analyzed. RESULTS: Over the study time frame, 48 EDS patients underwent surgery (mean age 52.6 ± 14.6 years; 48% females). Of these, 17 patients had vEDS and 31 patients had nEDS. Six patients (12.5%) underwent prior sternotomy. Urgent or emergent surgery was performed in 10 patients (20.8%). Aortic (vEDS 76.5% vs. nEDS 16.1%) and mitral procedures (vEDS 11.8% vs. nEDS 48.4%) were the two most common cardiovascular surgeries performed (p < .01 and p = .007, respectively). Cardiopulmonary bypass time (CPB) (165 ± 18 vs. 90 ± 13 min; p = .015) and aortic cross clamp times (140 ± 14 vs. 62 ± 10 min; p < .001) were longer for vEDS patients. There was 1 (2.1%) early and 7 (14.6%) late deaths; 6 among vEDS and 2 among nEDS patients. Survival at 5 (80% vs. 93%), 10 (45% vs. 84%) and 15 years (45% vs. 84%) was lower in patients with vEDS (p = .015 for each comparison). CONCLUSION: Cardiovascular surgeries are significantly more complex with longer bypass and cross clamp times for type IV vEDS compared to nEDS patients. Reduced overall survival underscores the complexity and fragility of vEDS patients.