Concomitant Mitral Regurgitation in Severe Aortic Stenosis　- Insights From the CURRENT AS Registry-2.

BACKGROUND: Data on concomitant mitral regurgitation (MR) in patients with severe aortic stenosis (AS) are scarce.Methods and Results: We investigated the risk of concomitant MR in patients with severe AS in the CURRENT AS Registry-2 according to initial treatment strategy (transcatheter aortic valve implantation [TAVI], surgical aortic valve replacement [SAVR], or conservative). Among 3,365 patients with severe AS, 384 (11.4%) had moderate/severe MR (TAVI: n=126/1,148; SAVR: n=68/591; conservative: n=190/1,626). The cumulative 3-year incidence for death or heart failure (HF) hospitalization was significantly higher in the moderate/severe than no/mild MR group in the entire population (54.6% vs. 34.3%, respectively; P<0.001) and for each treatment strategy (TAVI: 45.0% vs. 31.8% [P=0.006]; SAVR: 31.9% vs. 18.7% [P<0.001]; conservative: 67.8% vs. 41.6% [P<0.001]). The higher adjusted risk of moderate/severe MR relative to no/mild MR for death or HF hospitalization was not significant in the entire population (hazard ratio [HR] 1.15; 95% confidence interval [CI] 0.95-1.39; P=0.15); however, the risk was significant in the SAVR (HR 1.92; 95% CI 1.04-3.56; P=0.04) and conservative (HR 1.30; 95% CI 1.02-1.67; P=0.04) groups, but not in the TAVI group (HR 1.03; 95% CI 0.70-1.52; P=0.86), despite no significant interaction (Pinteraction=0.37). CONCLUSIONS: Moderate/severe MR was associated with a higher risk for death or HF hospitalization in the initial SAVR and conservative strategies, while the association was less pronounced in the initial TAVI strategy.

Difference in one-year late lumen loss between high- and low-dose paclitaxel-coated balloons for femoropopliteal disease.

The objective of the study is to investigate the difference in 1-year late lumen loss (LLL) between the high- (IN.PACT Admiral) and low-dose (Lutonix) paclitaxel-coated balloon (PCB). Although a recent randomized clinical trial demonstrated no difference in efficacy endpoint between high- and low-dose PCB, it remains unclear whether high-dose PCB was superior to low-dose PCB in actual clinical practice. We enrolled 64 patients with 67 de novo femoropopliteal lesions who underwent PCB angioplasty at Kokura Memorial Hospital from May 2014 to March 2020 and subsequent follow-up angiography after 1 year. The primary endpoint was 1-year LLL, whereas the secondary endpoints were binary restenosis and clinically driven target lesion revascularization (CD-TLR) after 1 year. The high- and low-dose PCB groups had 45 and 22 lesions, respectively. Although the low-dose PCB group had higher rates of coronary artery disease, hemodialysis, and chronic limb-threatening ischemia than the high-dose PCB group, the latter had a longer lesion length and more lesions with a TASC classification C or D than the former. The high-dose PCB group had a significantly lower LLL than the low-dose PCB group (0.40 ± 1.05 vs. 1.19 ± 1.03 mm; P = 0.003, respectively). Moreover, the high-dose PCB group had significantly lower rates of binary restenosis at 1 year than the low-dose PCB group (22.2% vs. 50.0%; P = 0.02, respectively). Moreover, negative LLL was only observed in the high-dose PCB group (33.3% vs. 0%, P = 0.005). The high-dose PCB group had a significantly lower LLL than the low-dose PCB group.

Rationale, Design, and Baseline Characteristics of the CURRENT AS Registry-2.

BACKGROUND: There is scarce data evaluating the current practice pattern and clinical outcomes for patients with severe aortic stenosis (AS), including both those who underwent surgical aortic valve replacement (SAVR) or transcatheter aortic valve implantation (TAVI) and those who were managed conservatively in the TAVI era.Methods and Results: The Contemporary outcomes after sURgery and medical tREatmeNT in patients with severe Aortic Stenosis (CURRENT AS) Registry-2 is a prospective, physician-initiated, multicenter registry enrolling consecutive patients who were diagnosed with severe AS between April 2018 and December 2020 among 21 centers in Japan. The rationale for the prospective enrollment was to standardize the assessment of symptomatic status, echocardiographic evaluation, and other recommended diagnostic examinations such as computed tomography and measurement of B-type natriuretic peptide. Moreover, the schedule of clinical and echocardiographic follow up was prospectively defined and strongly recommended for patients who were managed conservatively. The entire study population consisted of 3,394 patients (mean age: 81.6 years and women: 60%). Etiology of AS was degenerative in 90% of patients. AS-related symptoms were present in 60% of patients; these were most often heart failure symptoms. The prevalence of high- and low-gradient AS was 58% and 42%, respectively, with classical and paradoxical low-flow low-gradient AS in 4.6% and 6.7%, respectively. CONCLUSIONS: The CURRENT AS Registry-2 might be large and meticulous enough to determine the appropriate timing of intervention for patients with severe AS in contemporary clinical practice.

Mid-term impact of underfilling and overfilling of the SAPIEN 3 balloon-expandable transcatheter aortic valve implantation on mortality and valve function.

At present, underfilling or overfilling the volume of the balloon-expandable transcatheter heart valve (THV) is generally utilized in transcatheter aortic valve implantation (TAVI). However, no research has assessed the clinical impact of filling volume variations of the current-generation SAPIEN 3 THV. We analyzed the clinical data of 331 patients who underwent TAVI with SAPIEN 3 at our institution. Post-procedural echocardiographic and multidetector computed tomography (MDCT) scan data and 3-year prognoses according to each filling volume were assessed. The procedural outcomes and 3-year mortality rates were comparable among the underfilling, nominal filling, and overfilling groups. For all THV sizes, the THV area evaluated on post-procedural MDCT scan increased stepwise along with an elevated filling volume, thereby covering a wide range of native annulus area. Compared with patients in the nominal filling and overfilling groups, those with 23-mm THVs in the underfilling group had a smaller effective orifice area (EOA) (1.38 [IQR: 1.18-1.56] vs. 1.57 [IQR: 1.41-1.84] vs. 1.58 [IQR: 1.45-1.71] cm2, P = 0.02) and a higher mean transvalvular gradient (13.6 [IQR: 11.0-15.7] vs. 12.1 [IQR: 9.0-14.9] vs. 12.0 [IQR: 8.1-14.8] cm2, P = 0.04). In conclusion, by adjusting the filling volume of SAPIEN 3 using THV with limited sizes, continuously distributed native annulus areas were covered. The underfilling implantation technique had a minimal negative effect on the valve function of 23-mm THVs only. In the entire cohort, the filling volume variations did not affect the mid-term prognosis negatively.

Impact of serum cholinesterase on calcified nodules in patients with stable coronary artery disease.

BACKGROUND: Calcified nodules (CNs) are an advanced stage of coronary calcification that can have significant clinical implications. We investigated factors associated with CNs, the etiology of which is not fully understood. METHODS: We retrospectively evaluated 619 patients with stable coronary artery disease who underwent intravascular ultrasound (IVUS)-guided percutaneous coronary intervention (PCI). CNs in the culprit lesion were evaluated via IVUS, and all-cause mortality and major cardiovascular and cerebrovascular events (MACCEs) were compared between the CN and non-CN groups. RESULTS: The CN group (n = 40 patients) had a significantly lower survival rate and a higher incidence of MACCE than the non-CN group (P = 0.020 and P < 0.001, respectively). Multivariate logistic regression analysis models revealed that chronic kidney disease and serum cholinesterase (ChE) level were associated with CN formation [odds ratio (OR): 3.15, 95% confidence interval (CI): 1.30-7.69, P = 0.001 and OR: 0.94, 95% CI: 0.88-0.99, P = 0.042]. The optimal cutoff of serum ChE level as per the receiver operating characteristic curve was 309 units/l (Area under the curve = 0.67, sensitivity = 93%, specificity = 40%, P = 0.001). The low-ChE group divided according to the optimal cutoff value showed significantly higher cumulative incidence of MACCEs after PCI than the high-ChE group as per Kaplan-Meier analysis. CONCLUSION: The presence of CNs is significantly associated with a poor prognosis and MACCE after PCI among patients with stable coronary artery disease. Serum ChE levels may affect CN formation.

Clinically significant incidental noncardiac findings on preprocedural computed tomography in patients with aortic stenosis undergoing aortic-valve replacement.

BACKGROUND: There is a scarcity of data on the prevalence of abnormal findings on preprocedural computed tomography (CT) before aortic valve replacement (AVR) in patients with aortic stenosis (AS). METHODS: Among consecutive 593 patients with severe AS who were planned to undergo AVR, we evaluated the prevalence of clinically significant incidental noncardiac findings on preprocedural CT. Clinically significant incidental noncardiac findings were defined as newly detected abnormalities that required therapy, consultation for expert, further investigation, or clinical follow-up. RESULTS: The mean age was 82.0 years and 39.5 % of the patients were men. Of those, 78.4 % of the patients were treated with transcatheter aortic valve implantation (TAVI) and 21.6 % of the patients were treated with surgical AVR (SAVR). There were 271 clinically significant incidental noncardiac findings in 227 patients (38.3 %) including 2.5 % of malignancy. The prevalence of clinically significant incidental noncardiac findings were higher in the TAVI group than in the SAVR group (40.2 % versus 31.3 %). The prevalence of clinically significant incidental noncardiac findings were lower in patients under 60 years of age (10.0 %) than in patients over 60 years of age (60-69 years: 40.0 %, 70-79 years: 34.3 %, 80-89 years: 39.7 %, and ≥90 years: 42.1 %). CONCLUSIONS: Clinically significant incidental noncardiac findings were newly identified on preprocedural CT in approximately 40 % of patients with severe AS undergoing AVR including 2.5 % of malignancy.

Cerebrovascular Disease Detected on Preprocedural Computed Tomography in Patients With Severe Aortic Stenosis Undergoing Aortic Valve Replacement.

BACKGROUND: There is a scarcity of data on the prevalence and clinical impact of cerebrovascular disease detected on preprocedural computed tomography (CT) before aortic valve replacement (AVR) in patients with severe aortic stenosis. METHODS AND RESULTS: Among patients with severe aortic stenosis undergoing AVR, the authors compared clinical outcomes between patients with and without cerebrovascular disease detected on preprocedural CT, which was defined as chronic brain infarction or hemorrhage. The primary outcome measure in this study was a composite of all-cause death or stroke. Among 567 study patients, 200 patients (35.3%) had cerebrovascular disease on preprocedural CT. Among 200 patients with cerebrovascular disease on preprocedural CT, only 28.5% of patients had a clinical history of symptomatic stroke. The cumulative 3-year incidence of death or stroke was higher in patients with cerebrovascular disease on preprocedural CT than in those without cerebrovascular disease on preprocedural CT (40.7% versus 24.1%, log-rank P<0.001). After adjusting for confounders, the higher risk of patients with cerebrovascular disease on preprocedural CT relative to those without remained significant for death or stroke (hazard ratio [HR], 1.42 [95% CI, 1.02-1.98]; P=0.04). Among 200 patients with cerebrovascular disease on preprocedural CT, patients with prior symptomatic stroke compared with those without were not associated with higher adjusted risk for death or stroke (HR, 1.18 [95% CI, 0.72-1.94]; P=0.52). CONCLUSIONS: Among patients with severe aortic stenosis undergoing AVR, a substantial proportion had cerebrovascular disease on preprocedural CT, with a clinical history of symptomatic stroke in one-fourth of patients. Regardless of history of symptomatic stroke, patients with cerebrovascular disease on preprocedural CT had worse clinical outcomes compared with those without cerebrovascular disease on preprocedural CT.

Impact of Transjugular Intracardiac Echocardiography-Guided Self-Expandable Transcatheter Aortic Valve Implantation on Reduction of Conduction Disturbances.

BACKGROUND: A high permanent pacemaker implantation (PPI) risk remains a concern of self-expandable transcatheter aortic valve implantation, despite the continued improvements in implantation methodology. We aimed to assess the impact of real-time direct visualization of the membranous septum using transjugular intracardiac echocardiography (ICE) during transcatheter aortic valve implantation on reducing the rates of conduction disturbances including the need for PPI. METHODS: Consecutive patients treated with Evolut R and Evolut PRO/PRO+ from February 2017 to September 2022 were included in this study. We compared outcomes between the conventional implantation method using the 3-cusps view (3 cusps without ICE group), the recent method using cusp-overlap view (cusp overlap without ICE group), and our novel method using ICE (cusp overlap with ICE group). RESULTS: Of the 446 patients eligible for analysis, 211 (47.3%) were categorized as the 3 cusps without ICE group, 129 (28.9%) were in the cusp overlap without ICE group, and 106 (23.8%) comprised the cusp overlap with ICE group. Compared with the 3 cusps without ICE group, the cusp overlap without ICE group had a smaller implantation depth (2.2 [interquartile range, 1.0-3.5] mm versus 4.3 [interquartile range, 3.3-5.4] mm; P<0.001) and lower 30-day PPI rates (7.0% versus 14.2%; P=0.035). Compared with the cusp overlap without ICE group, the cusp overlap with ICE group had lower 30-day PPI rates (0.9%; P=0.014), albeit with comparable implantation depths (1.9 [interquartile range, 0.9-2.9] mm; P=0.150). Multivariable analysis showed that our novel method using ICE with the cusp-overlap view was independently associated with a 30-day PPI rate reduction. There were no group differences in 30-day all-cause mortality (1.4% versus 1.6% versus 0%; P=0.608). CONCLUSIONS: Our novel implantation method using transjugular ICE, which enable real-time direct visualization of the membranous septum, achieved a predictably high position of prostheses, resulting in a substantial reduction of conduction disturbances requiring PPI after transcatheter aortic valve implantation.

Assessing Potential Risks of Future Redo Transcatheter Aortic Valve Replacement in Asian Patients.

Background: In the Asian cohort, data are limited on the risk for coronary obstruction due to sinus of Valsalva (SOV) sequestration in redo transcatheter aortic valve replacement (TAVR) procedures. Objectives: The aim of this study was to assess the potential risk for coronary obstruction in simulated redo TAVR in Asian patients. Methods: Post-TAVR computed tomographic data from 788 patients who received balloon-expandable (BE) SAPIEN 3 transcatheter aortic valves (TAVs) and 334 patients who received self-expanding (SE) Evolut R or Evolut PRO TAVs were analyzed. The risk for coronary obstruction due to SOV sequestration in redo TAVR, defined as the TAV commissure level above the sinotubular junction (STJ) and a TAV-to-STJ distance <2.0 mm in each coronary sinus, was retrospectively evaluated. Results: The potential risks for coronary obstruction due to SOV sequestration at 1 or both coronary arteries were identified in 52.1% of the BE TAV group and 71.3% of the SE TAV group (P < 0.001). After adjusting for multiple covariates, STJ diameter, STJ height, TAV oversizing degree by area, and implantation depth were independently associated with SOV sequestration risk in the BE TAV group, whereas STJ diameter and implantation depth were independently associated with SOV sequestration risk in the SE TAV group. Conclusions: Coronary obstruction due to SOV sequestration in redo TAVR may occur in a substantial number of Asian patients. This finding suggests the importance of considering the structural feasibility of future redo TAVR when implanting the first TAV, especially in Asian patients with long life expectancy.

Long-term prognostic value of the H2FPEF score in patients undergoing transcatheter aortic valve implantation.

AIMS: A considerable proportion of candidates for transcatheter aortic valve implantation (TAVI) have underlying heart failure (HF) with preserved ejection fraction (HFpEF), which can be challenging for diagnosis because significant valvular heart disease should be excluded before diagnosing HFpEF. This study investigated the long-term prognostic value of the pre-procedural H2FPEF score in patients with preserved ejection fraction (EF) undergoing TAVI. METHODS AND RESULTS: Patients who underwent TAVI between October 2013 and May 2017 were enrolled from the Optimized CathEter vAlvular iNtervention-Transcatheter Aortic Valve Implantation Japanese multicentre registry. After excluding 914 patients, 1674 patients with preserved EF ≥ 50% (median age: 85 years, 72% female) were selected for calculation of the H2FPEF score and were dichotomized into two groups: the low H2FPEF score [0-5 points; n = 1399 (83.6%)] group and the high H2FPEF score [6-9 points; n = 275 (16.4%)] group. Patients with high H2FPEF scores were associated with a higher prevalence of New York Heart Association Functional Class III/IV (59.3% vs. 43.7%, P < 0.001), diabetes (24.4% vs. 18.5%, P = 0.03), and paradoxical low-flow, low-gradient aortic stenosis (15.9% vs. 6.2%, P < 0.001). These patients showed worse prognoses than those with low H2FPEF scores regarding the cumulative 2 year all-cause mortality (26.3% vs. 15.5%, log-rank P < 0.001), cardiovascular mortality (10.5% vs. 5.4%, log-rank P < 0.001), HF hospitalization (16.2% vs. 6.7%, log-rank P < 0.001), and the composite endpoint of cardiovascular mortality and HF hospitalization (23.8% vs. 10.8%, log-rank P < 0.001). After adjustment for several confounders, the high H2FPEF scores were independently associated with increased risk for all-cause mortality [adjusted hazard ratio (HR), 1.48; 95% confidence interval (CI), 1.09-2.00; P = 0.011] and for the composite endpoint of cardiovascular mortality and HF hospitalization (adjusted HR, 1.95; 95% CI, 1.38-2.74; P < 0.001). Subgroup analysis confirmed the excess risk of high H2FPEF scores relative to low H2FPEF scores for the composite endpoint of cardiovascular mortality and HF hospitalization increased with a lower Society of Thoracic Surgeons (STS) score (STS score <8%: adjusted HR, 2.40; 95% CI, 1.50-3.85; P < 0.001; STS score ≥8%: adjusted HR, 1.34; 95% CI, 0.79-2.28; P = 0.28; Pinteraction = 0.030). CONCLUSIONS: The H2FPEF score is useful for predicting long-term adverse outcomes after TAVI, including all-cause mortality, cardiovascular mortality, and HF hospitalization for patients with preserved EF. More aggressive interventions targeting HFpEF in addition to the TAVI procedure might be relevant in patients with high H2FPEF scores, particularly in those with a lower surgical risk.

In-hospital outcomes after SAVR or TAVI in patients with severe aortic stenosis.

There were no data comparing the in-hospital outcomes after transcatheter aortic valve implantation (TAVI) with those after surgical aortic valve replacement (SAVR) in Japan. Among consecutive patients with severe AS between April 2018 and December 2020 in the CURRENT AS Registry-2, we identified 1714 patients who underwent aortic valve replacement (TAVI group: 1134 patients, and SAVR group: 580 patients). Patients in the TAVI group were much older (84.4 versus 73.6 years, P < 0.001) and more often had comorbidities than those in the SAVR group. In-hospital death rate was numerically lower in the TAVI group than in the SAVR group (0.6% versus 2.2%). After excluding patients with dialysis, in-hospital death rate was very low and comparable in the TAVI and SAVR groups (0.6% versus 0.8%). The rates of major bleeding and new-onset atrial fibrillation during index hospitalization were higher after SAVR than after TAVI (72% versus 20%, and 26% versus 4.6%, respectively), while the rate of pacemaker implantation was higher after TAVI than after SAVR (8.1% versus 2.4%). Regarding the echocardiographic data at discharge, the prevalence of patient-prosthesis mismatch was lower in the TAVI group than in the SAVR group (moderate: 9.0% versus 26%, and severe: 2.6% versus 4.8%). In this real-world data in Japan, TAVI compared with SAVR was chosen in much older patients with more comorbidities with severe AS. In-hospital death rate was numerically lower in the TAVI group than in the SAVR group.

Three-dimensional transesophageal echocardiography-guided transseptal puncture for percutaneous mitral valve edge-to-edge repair post-percutaneous atrial septal defect closure.

Key Clinical Message: Three-dimensional multiplanar analysis and real-time three-dimensional guidance using transesophageal echocardiography can help to identify and access the ideal position for transseptal puncture even in the presence of atrial septal occluders. Abstract: Transseptal puncture (TSP) for the percutaneous mitral valve edge-to-edge repair (PMVR) after percutaneous atrial septal defect (ASD) closure is a rare and challenging issue. Here, we present a case illustrating the feasibility of real-time three-dimensional transesophageal echocardiographic guidance for TSP without ASD closure device injury.

Complement dependent TNFα production in neutrophil-like HL60 cells.

Neutrophils develop in the bone marrow (BM) from hematopoietic stem cells (HSCs) through a series of progenitor cells and mature neutrophils play a critical role in the human immune system. Previous studies revealed that tumor necrosis factor α (TNFα) produced by immature neutrophils contributes to HSCs development and vascular regeneration in the BM niche. However, the precise mechanism of TNFα production in immature neutrophils remains unclear. This study aims to assess the relationship between complement C3 activation and TNFα production from immature neutrophils. We investigated the regulatory mechanism of TNFα production by complement components in neutrophil-like HL60 cells. Flow cytometric analysis showed that C3a receptor (C3aR) and C3bi receptor (CR3, Mac-1, CD11b/CD18, integrin αMß2) are expressed on the surface of neutrophil-like HL60 cells. We found that zymosan-treated human serum leads to TNFα production in neutrophil-like HL60 cells, but not in human polymorphonuclear cells (PMNs). A C3-convertase inhibitor, compstatin suppresses TNFα production. These data suggest that the TNFα production is mediated by complement C3 activation. Furthermore, the TNFα production is enhanced by Ca2+ elevating agents, thapsigargin (TG), but is suppressed by treatment with Ca2+ chelators, EGTA, or BAPTA-AM. In addition, the soluble TNFα production is suppressed by treatment with immobilized-fibrinogen or -fibronectin. Thus, the TNFα production is enhanced by intracellular Ca2+ elevation and is negatively regulated by the interaction between the neutrophil-like HL60 cells and fibrinogen or fibronectin.

Prevalence and Prognostic Significance of Malnutrition in Older Japanese Adults at High Surgical Risk Undergoing Transcatheter Aortic Valve Implantation.

Background The usefulness of preprocedural nutritional status to stratify prognosis after transcatheter aortic valve implantation has been evaluated; however, the studies conducted so far have been relatively small and/or focused on a single nutritional index. This study sought to assess the prevalence and prognostic impact of malnutrition in patients with severe aortic stenosis undergoing transcatheter aortic valve implantation. Methods and Results We applied the Controlling Nutritional Status score, Geriatric Nutritional Risk Index, and Prognostic Nutritional Index to 1040 consecutive older Japanese patients at high surgical risk who underwent transcatheter aortic valve implantation. According to the Controlling Nutritional Status score, Geriatric Nutritional Risk Index, and Prognostic Nutritional Index, 16.6%, 60.5%, and 13.8% patients had moderate or severe malnutrition, respectively; 89.3% were at least mildly malnourished by at least 1 score. Worse nutritional status was associated with older age, lower body mass index, higher degree of frailty, worse symptoms and renal function, atrial fibrillation, and anemia. During a median follow-up of 986 days (interquartile range, 556-1402 days), 273 (26.3%) patients died. Compared with normal nutrition, malnutrition was associated with an increased risk for all-cause death (adjusted hazard ratio for moderate and severe malnutrition, respectively: 2.19 (95% CI, 1.45-3.31; P<0.001) and 6.13 (95% CI, 2.75-13.70; P<0.001) for the Controlling Nutritional Status score, 2.02 (95% CI, 1.36-3.02; P=0.001) and 3.24 (95% CI, 1.86-5.65; P<0.001) for the Geriatric Nutritional Risk Index, and 1.60 (95% CI, 1.06-2.39; P=0.024) and 2.32 (95% CI, 1.50-3.60; P<0.001) for the Prognostic Nutritional Index). Conclusions Malnutrition is common in patients undergoing transcatheter aortic valve implantation and is associated with increased mortality.

Vacuolar-type proton ATPase is required for maintenance of apicobasal polarity of embryonic visceral endoderm.

The endocytic compartments keep their interior acidic through the inward flow of protons and anions from the cytosol. Acidification is mediated by a proton pump known as vacuolar-type ATPase (V-ATPase) and transporters conferring anion conductance to the organellar membrane. In this study, we analysed the phenotype of mouse embryos lacking the V-ATPase c-subunit. The mutant embryos differentiated embryonic epithelial tissues, primitive endoderm, epiblast, and extraembryonic ectoderm; however, the organisation of these epithelia was severely affected. The apical-basal polarity in the visceral endoderm layer was not properly established in the mutant embryos, resulting in abnormal epithelial morphology. Thus, the function of V-ATPase is imperative for the establishment and/or maintenance of epithelial cell polarity, which is required for early embryogenesis.

Syk facilitates phagosome-lysosome fusion by regulating actin-remodeling in complement-mediated phagocytosis.

Effective phagocytosis is crucial for host defense against pathogens. Macrophages entrap pathogens into a phagosome and subsequently acidic lysosomes fuse to the phagosome. Previous studies showed the pivotal role of actin-remodeling mediated by phosphoinositide-related signaling in phagosome formation, but the mechanisms of phagosome-lysosome fusion remain unexplored. Here we show that in complement-mediated phagocytosis, phagosome-lysosome fusion requires the disappearance of F-actin structure surrounding the phagosome and a tyrosine kinase Syk plays a key role in this process. Using macrophage-like differentiated HL60 and Syk-knockout (Syk-KO) HL60 cells, we found that Syk-KO cells showed insufficient phagosome acidification caused by impaired fusion with lysosomes and permitted the survival of Candida albicans in complement-mediated phagocytosis. Phagosome tracking analysis showed that during phagosome internalization process, F-actin surrounding phagosomes disappeared in both parental and Syk-KO cells but this structure was reconstructed immediately only in Syk-KO cells. In addition, F-actin-stabilizing agent induced a similar impairment of phagosome-lysosome fusion. Collectively, Syk-derived signaling facilitates phagosome-lysosome fusion by regulating actin-remodeling.

Prostaglandin EP3 receptor superactivates adenylyl cyclase via the Gq/PLC/Ca2+ pathway in a lipid raft-dependent manner.

We previously demonstrated that prostaglandin EP3 receptor augments EP2-elicited cAMP formation in COS-7 cells in a G(i/o)-insensitive manner. The purpose of our current study was to identify the signaling pathways involved in EP3-induced augmentation of receptor-stimulated cAMP formation. The enhancing effect of EP3 receptor was irrespective of the C-terminal structure of the EP3 isoform. This EP3 action was abolished by treatment with inhibitors for phospholipase C and intracellular Ca(2+)-related signaling molecules such as U73122, staurosporine, 2-APB and SK&F 96365. Indeed, an EP3 agonist stimulated IP(3) formation and intracellular Ca(2+) mobilization, which was blocked by U73122, but not by pertussis toxin. The enhancing effect by EP3 on cAMP formation was mimicked by both a Ca(2+) ionophore and the activation of a typical G(q)-coupled receptor. Moreover, EP3 was exclusively localized to the raft fraction in COS-7 cells and EP3-elicited augmentation of cAMP formation was abolished by cholesterol depletion and introduction of a dominant negative caveolin-1 mutant. These results suggest that EP3 elicits adenylyl cyclase superactivation via G(q)/phospholipase C activation and intracellular Ca(2+) mobilization in a lipid raft microdomain-dependent manner.

KIF20A, highly expressed in immature hematopoietic cells, supports the growth of HL60 cell line.

A microtubule-associated motor protein, kinesin-like family member 20A (KIF20A; also called MKlp2) is required for cytokinesis and contributes to intracellular vesicular trafficking. KIF20A plays a critical role in the development of several cancers, but its role in blood cells and hematological malignancies have not been studied. In the present study, we focused on the role of KIF20A in hematopoietic cells and possible involvement in myeloid neoplasms. We found that human leukemia cell lines and normal bone marrow CD34-positive cells stimulated by growth factors, but not mature peripheral blood cells, exhibit high KIF20A expression. We further found that HL60 cells, which originally express a large amount of KIF20A, showed decreased KIF20A expression in parallel with both neutrophil-like and macrophage-like differentiation-induction. KIF20A-knockdown using a lentivirus shRNA transfection system led to partial cell cycle arrest at the G2/M phase and frequent appearance of multinucleated cells. Treatment with a KIF20A-selective inhibitor, paprotrain enhanced the multinuclearity of KIF20A-knockdown cell clones and suppressed growth. The present study contributes to our understanding of the role of KIF20A in blood cells and leukemia cells in particular.