Citations

Angiotensin II (Ang II) type I receptor (ATR 1 ) trafficking into caveolae is essential for Ang II signaling, which is known to be abnormal in diabetic vessels. We have shown that the large conductance Ca 2+ actviated K + (BK) channels are also targeted to caveolae in vascular cells. The potential interaction between Ang II signaling and BK channel function in normal and diabetic vessels is unknown. Using whole-cell patch clamp recordings and molecular biology techniques, we examined the mechanisms through which caveolae targeting facilitates the regulation of BK channels by Ang II signaling. We found that in cultured human coronary arterial smooth muscle cells (CASMC) and in freshly isolated rat CASMC, BK channels, ATR 1 , and Src-family protein tyrosine kinases (Src-PTK) were colocalized and enriched in the low buoyant density, caveolae-rich fractions. 2 μM Ang II inhibited BK channel activity by ∼50% in rat and human CASMC and these effects were completely abolished by 2 μM Losartan (a selective ATR 1 inhibitor), 10 μM PP2 (a selective Src-PTK inhibitor), and by caveolin-1 (cav-1) knockdown using 60 nM siRNA. Similar results were obtained in HEK293 cells coexpressing hSlo, BK-β 1 subunit, ATR 1 , cav-1, and Src-PTK, indicating that inhibition of BK channels by Ang II was mediated through ATR 1 activation of Src-PTK and the integrity of caveolae is critical for Ang II signaling. Culturing human CASMC with high glucose (HG, 22 mM) enhanced Ang II-mediated BK channel inhibition (78.8±16.8% vs. 54.5±15.7% in 5 mM glucose, n=3, p<0.05). Analysis of ATR 1 , Src-PTK, and BK channel distribution by sucrose gradient fractionation and by co-immunoprecipitation with anti-cav-1 antibodies showed that expression of ATR 1 and Src-PTK were up-regulated in human CASMC cultured in HG and in CASMC from streptozotocin-induced diabetic rats. Total BK channel protein in these cells was diminished, but the amount of BK channels co-immunoprecipitated with anti-cav-1 antibody was increased, suggesting increased caveolae targeting of BK channels in diabetes, which leads to enhanced Ang II-mediated BK channel inhibition. These results indicate that Ang II-BK channel interaction is critically dependent upon caveolae targeting under normal conditions and in disease states such as diabetes.

Introduction: Left atrial appendage (LAA) flow depends largely on left ventricular compliance and may play a role in mediating the regulation of left atrial volume-pressure relationships. Hypothesis: We hypothesize that LAA emptying flow velocity (LAAEV) is a predictive factor of long-term outcomes (e.g. recurrent AF, stroke, and survival) after cardioversion for non-valvular AF. Method: We identified 3,251 consecutive patients with non-valvular AF who underwent successful TEE-guided electrical cardioversion (ECV) at our institution between May 2000 and March 2012. Successful ECV was defined as sinus rhythm at time of discharge from the cardioversion unit. Patients were monitored following their ECV procedure for first documentation of recurrent AF, stroke or death. Multivariate Cox proportional hazards models were used to identify independent predictors of long-term outcomes. Patients with >= moderate valvular regurgitation or stenosis were excluded. Results: Among the 3,251 patients who were successfully cardioverted to sinus rhythm, the mean (±SD) LAAEV was 38.43±23 cm/s and the median was 33 cm/s, (interquartile range [IQR], 20-50). Patients with LAAEV <=33 cm/s had higher CHA 2 DS 2 -VASc score (2.6±1.2 vs. 1.9±1.3, P =.009), larger LAVI (52.0±20.9 cc/m 2 vs. 43.3±13.6 cc/m 2 , P <.001) than those with LAAEV >33 cm/s. Pre or post-procedure antiarrhythmic drug use was similar between the two groups. During 1-year follow-up, patients with LAAEV <=33 cm/s had significantly higher rate of AF recurrence than those with LAAEV >33 cm/s (55% vs 45%, P <.001). Likewise, during a mean follow-up of 4.9±3.6 years, similar patterns in 5-year rates were observed for first recurrence of AF (81% vs 73%, P <.001), stroke (7% vs 4%, P =.003) and mortality (31% vs 23%, P <.001) for LAAEV <=33 vs > 33cm/s, respectively. Stepwise multivariate Cox regression analysis revealed that LAAEV <=33 cm/s, age, CHA 2 DS 2 -VASc score were independent predictors of AF recurrence, stroke and mortality. Conclusions: LAA emptying flow velocity is an effective and convenient method for risk stratification of patients undergoing cardioversion for AF. Our data showed that patients with reduced LAAEV have an increased risk for AF recurrence, stroke and death following electrical cardioversion.

GTP Cyclohydrolase‐I (GTPCHI) is the rate‐limiting enzyme for the synthesis of tetrahydrobiopterin (BH4), which is an essential cofactor for the enzymatic activity of endothelial nitric oxide synthase (eNOS). To better understand the role of GTPCHI in maintaining eNOS function, we analyzed GTPCHI subcellular localization and activity in human umbilical cord vein endothelial cells (HUVEC). Western blot analysis showed similar levels of GTPCHI protein expression in both Triton X‐100 ‐soluble and ‐insoluble fractions, suggesting membrane targeting of GTPCHI. Further analysis of membrane‐associated proteins using sucrose gradient ultracentrifugation revealed that GTPCHI protein expression was localized to caveolar microdomains along with caveolin‐1(Cav‐1) and eNOS. HPLC analysis for GTPCHI activity confirmed the localization of GTPCHI to caveolar microdomains as well as golgi‐associated membrane fractions. Using an in vitro GST pull‐down assay with recombinantGST‐caveolin‐1 fusion protein incubated with the lysate of HUVEC, we demonstrated specific binding of GTPCHI to caveolin‐1. To determine if caveolin‐1 may regulate GTPCHI activity in vivo, we analyzed aortas from Cav‐1 −/ − mice and were able to detect a two‐fold increase in BH4 production and GTPCHI activity in the aortas of Cav‐1 −/ − mice when compared to eNOS −/ − mice or wild type mice. These studies reveal that BH4 synthesis can occur in specialized membrane compartments in close proximity to eNOS and may be subjected to posttranslational regulation by caveolin‐1 which has an inhibitory effect on GTPCHI enzyme activity. Funded by NIH grant HL‐53524.

Epoxyeicosatrienoic acids (EETs) potently and stereospecifically activate the cardiac ATP‐sensitive K + (Kir6.2) channels by reducing channel sensitivity to ATP. In this study, we determined the effects of EETs on the binding of ATP to Kir6.2 by using 2,4,6‐trinitrophenyl (TNP)‐ATP, which increases fluorescence emission >8‐fold with binding to protein, and by using GST fusion protein containing Kir6.2. TNP‐ATP binds to the GST fusion protein containing the Kir6.2C‐terminus but not to that with the Kir6.2N‐terminus. Binding of TNP‐ATP to GST‐Kir6.2C (K D 5.0 μ M) was unchanged by 5 μ M 11,12‐EET (K D 4.4 μ M) but B max was reduced by half, suggesting non‐competitive inhibition of ATP binding. The effect of 11,12‐EET was dose‐dependent and 8,9‐ and 14,15‐EETs were equipotent inhibitors of TNP‐ATP binding to GST‐Kir6.2C. Arachidonic acid and 11,12‐DHET were not effective inhibitors, whereas the methyl ester of 11,12‐EET was as effective as 11,12‐EET in inhibiting TNP‐ATP binding to GST‐Kir6.2C. R195A mutation in GST‐Kir6.2C abolished the EET effects on TNP‐ATP binding, while the K185A mutation, which reduced ATP binding to Kir6.2, had no effect. These findings are consistent with previous patch clamp results that showed the epoxide group in EET and R195 in Kir6.2 are critical for K ATP channel activation by EET. We conclude that EETs bind to the C‐terminus of the K ATP channel and allosterically alter the ATP binding site, reducing the channel sensitivity to ATP.

This poster presents an expert consensus position on the current controversy regarding calcium supplement use and risk of myocardial infarction. In November 2011, The CRN Foundation with the support of the National Osteoporosis Foundation and National Bone Health Alliance convened an expert panel of scientists from the nutrition and medical communities to review the total body of evidence and specifically studies relating calcium supplements to an increased risk of myocardial infarction. Because of the many data limitations present in the supporting studies relating calcium supplements to an increased risk of myocardial infarction and contrast to the large majority of the literature accompanied with recent reanalyses of the data, no change in prescribing practice is currently recommended. Prescribers should consider the potential benefits of calcium supplements for prevention of osteoporotic fractures based on an individual's personal dietary intake. Positive outcomes of calcium supplementation such as prevention of obesity and colorectal cancers may also carry weight in support of calcium supplements when individual dietary intakes are inadequate to meet IOM recommendations.

We examined the role of H 2 S on shear stress‐mediated dilation of isolated mouse coronary arteries. Na 2 S produced concentration‐dependent dilation in these vessels, which was significantly inhibited by iberiotoxin. BK currents in mouse coronary smooth muscle cells were directly activated by Na 2 S, suggesting H 2 S produced vasodilation through BK channel activation. Using a pressure servo controller system, freshly isolated mouse coronary arteries were subjected to physiological levels of shear stress (1 to 25 dynes/cm 2 ), producing graded dilatory responses, but such effects were diminished in the presence of 100 μM Na 2 S. Pre‐incubation with the cystathionine γ‐lyase inhibitor, D,L‐propargylglycine (PPG), resulted in a paradoxical augmentation of shear stress‐mediated vasodilation. However, in the presence of L‐NAME or in coronary arteries from eNOS knockout mice, PPG inhibited shear stress‐mediated vasodilation, suggesting an interaction between NO and H 2 S signaling. Using cultured bovine aortic endothelial cells, eNOS activity was inhibited by Na 2 S. These results suggest that both NO and H 2 S are important shear stress‐mediated vasodilators in mouse coronary arteries but there is a complex interaction between these two signaling pathways that results in paradoxical vasoconstrictive effects of H 2 S through inhibition of NO generation. (Supported by grants from NIH)

PURPOSE This study assessed the joint association of pathogenic variants (PVs) in breast cancer (BC) predisposition genes and polygenic risk scores (PRS) with BC in the general population. METHODS A total of 26,798 non-Hispanic white BC cases and 26,127 controls from predominately population-based studies in the Cancer Risk Estimates Related to Susceptibility consortium were evaluated for PVs in BRCA1, BRCA2, ATM, CHEK2, PALB2, BARD1, BRIP1, CDH1, and NF1. PRS based on 105 common variants were created using effect estimates from BC genome-wide association studies; the performance of an overall BC PRS and estrogen receptor–specific PRS were evaluated. The odds of BC based on the PVs and PRS were estimated using penalized logistic regression. The results were combined with age-specific incidence rates to estimate 5-year and lifetime absolute risks of BC across percentiles of PRS by PV status and first-degree family history of BC. RESULTS The estimated lifetime risks of BC among general-population noncarriers, based on 10th and 90th percentiles of PRS, were 9.1%-23.9% and 6.7%-18.2% for women with or without first-degree relatives with BC, respectively. Taking PRS into account, more than 95% of BRCA1, BRCA2, and PALB2 carriers had > 20% lifetime risks of BC, whereas, respectively, 52.5% and 69.7% of ATM and CHEK2 carriers without first-degree relatives with BC, and 78.8% and 89.9% of those with a first-degree relative with BC had > 20% risk. CONCLUSION PRS facilitates personalization of BC risk among carriers of PVs in predisposition genes. Incorporating PRS into BC risk estimation may help identify > 30% of CHEK2 and nearly half of ATM carriers below the 20% lifetime risk threshold, suggesting the addition of PRS may prevent overscreening and enable more personalized risk management approaches.

11536 Background: Ripretinib is a switch-control tyrosine kinase inhibitor that broadly inhibits KIT and PDGFRA kinase signaling. In the INVICTUS study (NCT03353753), patients with advanced GIST (≥4th-line) receiving ripretinib had a median progression-free survival (mPFS) of 6.3 months vs 1.0 month for patients receiving placebo (HR = 0.15, p <0.0001). In an earlier phase 1 dose escalation study, the maximum tolerated dose was not reached with doses up to 200 mg twice daily (BID). Here, we report efficacy, safety, and pharmacokinetic data for IPDE patients initially randomized to ripretinib from the phase 3 INVICTUS study based on data as of 10 Aug 2020. Methods: A total of 129 patients were randomized 2:1 to ripretinib 150 mg once daily (QD; n = 85) or placebo (n = 44). Patients receiving ripretinib 150 mg QD who had progressive disease (PD) as assessed by blinded independent central review (BICR) using mRECIST were given the option for IPDE to 150 mg BID. Tumor response assessments were performed every 28 days for 4 cycles and every 56 days thereafter (including after IPDE). The primary endpoint was PFS. For this exploratory analysis, PFS1 for IPDE patients was defined as the time from randomization to PD; PFS2 for IPDE patients was the time from the first dose of ripretinib 150 mg BID to PD or death. PFS1 and PFS2 were based on BICR. Results: Of 85 patients treated with ripretinib 150 mg QD, 43 patients with BICR PD dose escalated to 150 mg BID. Baseline characteristics of IPDE patients at time of study entry were similar to those observed in the original ripretinib QD arm and similar to the 22 patients with BICR PD who either remained on 150 mg QD or discontinued treatment. IPDE patients had a mPFS1 of 4.6 months (95% CI, 2.7–6.4) and a mPFS2 of 3.7 months (95% CI, 3.1–5.3); mPFS2/mPFS1=80%. The IPDE dosing period was well tolerated without the emergence of new safety concerns. The most common new or worsening (unchanged or improving grades not included) treatment-emergent adverse event (TEAE) for IPDE patients during the BID period was abdominal pain (30.2% all grades and 7% Grade 3–4 vs 41.9% and 4.7% in QD period, respectively). The most common Grade 3–4 TEAE in the BID period was anemia (14% vs 2.3% in QD period). IPDE from QD to BID resulted in an approximately 2-fold increase in the steady state trough concentration. Conclusions: Similar to the phase 1 study wherein IPDE to 150 mg BID following PD provided clinical benefit with a mPFS1 of 5.5 months and mPFS2 of 4.6 months (mPFS2/mPFS1=84%) for patients with ≥4th-line GIST, these analyses from INVICTUS indicate that IPDE to ripretinib 150 mg BID can also provide additional meaningful clinical benefit and a similar tolerability profile to the 150 mg QD regimen for patients with ≥4th-line GIST that progressed following treatment with ripretinib 150 mg QD. Clinical trial information: NCT03353753.

Background: Pouchitis is the most common long-term complication after ileal pouch-anal anastomosis in patients with ulcerative colitis. Those with ≥3 episodes of pouchitis/year and symptoms despite antibiotics are considered to have chronic antibiotic refractory pouchitis (CARP). While several agents including probiotics, steroids and immunomodulators have been used, treatment of CARP remains challenging. We conducted a systematic review and meta-analysis evaluating the safety and efficacy of various biological agents in treatment of CARP. Methods: Multiple databases were searched through June 2020 for studies that reported the efficacy and safety of biological therapy including antitumor necrosis factor-alpha agents [infliximab (IFX) and adalimumab (ADA)], vedolizumab (VDZ), and ustekinumab in CARP. We excluded studies on Crohn’s like and/or other inflammatory complications of the pouch. Meta-analysis was performed to calculate pooled rates of clinical as well as endoscopic improvement and remission. Results: We included 15 studies with 311 patients in our final analysis. Ninety-two patients were treated with IFX, 42 with ADA, 144 with VDZ and 33 with ustekinumab. Pooled rate of clinical improvement was 71.4%, 58.2%, 47.9% and clinical remission was 65.7%, 31%, 47.4% with IFX, ADA, and VDZ, respectively. Pooled rate of endoscopic improvement was achieved in 61.2% patients treated with VDZ while endoscopic remission was achieved in 70.3% patients treated with IFX. Adverse events were reported in 3.9% patients. Conclusion: Biologic therapy is safe and effective in the treatment of CARP.