RNA-binding protein LIN28A upregulates transcription factor HIF1α by posttranscriptional regulation via direct binding to UGAU motifs.

Hypoxia-inducible factor 1α (HIF1α) is a transcription factor that regulates angiogenesis under hypoxic conditions. To investigate the posttranscriptional regulatory mechanism of HIF1α, we performed a cell-based screening to reveal potential cis-elements and the regulatory RNA-binding proteins that act as trans-factors. We found that LIN28A promoted HIF1α protein expression independently of the downregulation of microRNA let-7, which is also directly mediated by LIN28A. Transcriptome analysis and evaluation of RNA stability using RNA-seq and SLAM-seq analyses, respectively, revealed that LIN28A upregulates HIF1A expression via mRNA stabilization. To investigate the physical association of LIN28A with HIF1A mRNA, we performed enhanced crosslinking immunoprecipitation in 293FT cells and integrally analyzed the transcriptome. We observed that LIN28A associates with HIF1A mRNA via its cis-element motif "UGAU". The "UGAU" motifs are recognized by the cold shock domain of LIN28A, and the introduction of a loss-of-function mutation to the cold shock domain diminished the upregulatory activities performed by LIN28A. Finally, the microvessel density assay showed that the expression of LIN28A promoted angiogenesis in vivo. In conclusion, our study elucidated the role of LIN28A in enhancing the HIF1α axis at the posttranscription layer.

The tRNA pseudouridine synthase TruB1 regulates the maturation of let-7 miRNA.

Let-7 is an evolutionary conserved microRNA that mediates post-transcriptional gene silencing to regulate a wide range of biological processes, including development, differentiation, and tumor suppression. Let-7 biogenesis is tightly regulated by several RNA-binding proteins, including Lin28A/B, which represses let-7 maturation. To identify new regulators of let-7, we devised a cell-based functional screen of RNA-binding proteins using a let-7 sensor luciferase reporter and identified the tRNA pseudouridine synthase, TruB1. TruB1 enhanced maturation specifically of let-7 family members. Rather than inducing pseudouridylation of the miRNAs, high-throughput sequencing crosslinking immunoprecipitation (HITS-CLIP) and biochemical analyses revealed direct binding between endogenous TruB1 and the stem-loop structure of pri-let-7, which also binds Lin28A/B. TruB1 selectively enhanced the interaction between pri-let-7 and the microprocessor DGCR8, which mediates miRNA maturation. Finally, TruB1 suppressed cell proliferation, which was mediated in part by let-7. Altogether, we reveal an unexpected function for TruB1 in promoting let-7 maturation.

A Runx1-enhancer Element eR1 Identified Lineage Restricted Mammary Luminal Stem Cells.

Mammary gland homeostasis is maintained by adult tissue stem-progenitor cells residing within the luminal and basal epithelia. Dysregulation of mammary stem cells is a key mechanism for cancer development. However, stem cell characterization is challenging because reporter models using cell-specific promoters do not fully recapitulate the mammary stem cell populations. We previously found that a 270-basepair Runx1 enhancer element, named eR1, marked stem cells in the blood and stomach. Here, we identified eR1 activity in a rare subpopulation of the ERα-negative luminal epithelium in mouse mammary glands. Lineage-tracing using an eR1-CreERT2 mouse model revealed that eR1+ luminal cells generated the entire luminal lineage and milk-secreting alveoli-eR1 therefore specifically marks lineage-restricted luminal stem cells. eR1-targeted-conditional knockout of Runx1 led to the expansion of luminal epithelial cells, accompanied by elevated ERα expression. Our findings demonstrate a definitive role for Runx1 in the regulation of the eR1-positive luminal stem cell proliferation during mammary homeostasis. Our findings identify a mechanistic link for Runx1 in stem cell proliferation and its dysregulation in breast cancer. Runx1 inactivation is therefore likely to be an early hit in the cell-of-origin of ERα+ luminal type breast cancer.

Impact of calcification on clinical outcomes after drug-coated balloon angioplasty for superficial femoral artery disease: Assessment using the peripheral artery calcification scoring system.

PURPOSE: To investigate whether the severity of calcification assessed by the peripheral artery calcification scoring system (PACSS) was associated with clinical outcomes of drug-coated balloon (DCB) angioplasty for femoropopliteal lesions. MATERIALS AND METHODS: We retrospectively analyzed 733 limbs with intermittent claudication of 626 patients, who underwent DCB angioplasty for de novo femoropopliteal lesions between January 2017 and February 2021 at seven cardiovascular centers in Japan. The patients were categorized using the PACSS classification (grades 0-4: no visible calcification of the target lesion, unilateral wall calcification < 5 cm, unilateral calcification ≥ 5 cm, bilateral wall calcification < 5 cm, and bilateral calcification ≥ 5 cm, respectively). The main outcome was primary patency at 1 year. The Cox proportional hazards analysis was used to explore whether the PACSS classification was an independent predictor of clinical outcomes. RESULTS: The distribution of PACSS was grade 0 in 38%, grade 1 in 17%, grade 2 in 7%, grade 3 in 16%, and grade 4 in 23%. The 1-year primary patency rates in these grades, respectively, were 88.2%, 89.3%, 71.9%, 96.5%, and 82.6%, respectively (p < 0.001). Multivariate analysis disclosed that PACSS grade 4 (hazard ratio: 1.82, 95% confidence interval 1.15-2.87, p = 0.010) was associated with restenosis. CONCLUSION: The PACSS grade 4 calcification was independently associated with poor clinical outcomes after DCB angioplasty for de novo femoropopliteal lesions.

Ten-year clinical outcomes from a randomized trial comparing new-generation everolimus-eluting stent versus first-generation Sirolimus-eluting stent: Results from the RESET extended study.

BACKGROUND: New-generation drug-eluting stents (DES) achieved technological innovations and reported clinical advantages as compared with first-generation DES in clinical trials with 3-5 years follow-up. However, detailed clinical outcome data in very long-term follow-up is still scarce. OBJECTIVES: To evaluate 10-year clinical outcomes after first- and new-generation DES implantation. METHODS: In this extende follow-up study of the RESET, which is a largest randomized trial comparing everolimus-eluting stent (EES) with Sirolimus-eluting stent (SES), the study population consisted of 2892 patients from 84 centers. The primary efficacy and safety endpoints were target lesion revascularization (TLR) and a composite of death or myocardial infarction (MI), respectively. Complete 10-year follow-up was achieved in 87.9% of patients. RESULTS: Cumulative 10-year incidences of TLR and non-TLR were not significantly different between EES and SES (13.9% vs. 15.7%, Log-rank p = 0.20, and 33.4% vs. 31.3%, Log-rank p = 0.30). The cumulative 10-year incidence of death/MI was also not significantly different between the groups (32.5% vs. 34.4%, Log-rank p = 0.18). Cumulative 10-year incidence of definite stent thrombosis was numerically lower in EES than in SES (1.0% vs. 1.7%, Log-rank p = 0.16). The lower risk of EES relative to SES was significant for a composite endpoint of target lesion failure (TLF: 19.6% vs. 24.9%, Log-rank p = 0.001) and target vessel failure (TVF: 26.7% vs. 31.4%, Log-rank p = 0.006). CONCLUSION: During 10-year of follow-up, the risks for primary efficacy and safety endpoints were not significantly different between new-generation EES and first-generation SES, although EES compared with SES was associated with a lower risk for composite endpoints such as TLF and TVF.

Prediction of Wound Recurrence in Patients With Chronic Limb-Threatening Ischemia Undergoing Endovascular Treatment.

PURPOSE: Chronic limb-threatening ischemia due to isolated below-the-knee lesions is a factor associated with wound recurrence. However, there is a lack of data regarding wound recurrence in such cases. This study aimed to determine the predictors of wound recurrence in patients with chronic limb-threatening ischemia undergoing endovascular treatment. PATIENTS AND METHODS: This was a single-center, retrospective, observational study. We enrolled 152 consecutive patients with chronic limb-threatening ischemia (172 limbs) who achieved complete wound healing after undergoing endovascular treatment for isolated below-the-knee lesions between February 2008 and December 2017. Of these, the wound had recurred in 56 limbs (33%), and we divided the patients into 2 groups based on wound recurrence. We evaluated the recurrence rate of chronic limb-threatening ischemia and predictors of wound recurrence. Wound recurrence was defined as recurrence of the wound within 2 years of complete wound healing. RESULTS: Patients' backgrounds were similar in both groups, including mean age (72±9 vs 72±11; p=0.76) and hemodialysis (43% vs 40%; p=0.66). Pre-pedal arch type 2 (52% vs 8%; p<0.01), infrapopliteal grade 4 of the Global Limb Anatomic Staging System (77% vs 59%; p=0.02), and Wound, Ischemia, and foot Infection criteria stage 4 (43% vs 28%; p=0.04) were more common in the wound recurrence group. Multivariate Cox proportional hazard analysis identified pre-pedal arch type 2 (hazard ratio, 5.28; 95% confidence interval, 3.08-9.10; p<0.01) and Wound, Ischemia, and foot Infection criteria stage 4 (hazard ratio, 1.98; 95% confidence interval, 1.15-3.36; p=0.01) as predictors of wound recurrence after complete wound healing. CONCLUSION: Pre-pedal arch type 2 and Wound, Ischemia, and foot Infection classification system stage 4 were associated with wound recurrence in patients with chronic limb-threatening ischemia who achieved complete wound healing after undergoing endovascular treatment for isolated below-the-knee lesions.

Drug-Coated Balloon for the Treatment of Small Vessel Coronary Artery Disease　- A Randomized Non-Inferiority Trial.

BACKGROUND: Drug-coated balloons (DCB) have shown promising results for the treatment of in-stent restenosis (ISR) and small vessel disease (SVD). However, data comparing the treatment efficacy of different DCBs are limited.Methods and Results: AGENT Japan is a prospective randomized controlled trial that compares the Agent balloon coated with a low-dose formulation of paclitaxel (2 µg/mm2) to the SeQuent Please paclitaxel-coated balloon (3 µg/mm2) for the treatment of SVD. Patients with target lesion length ≤28 mm and reference diameter between ≥2.00 and <3.00 mm were randomized 2 : 1 for treatment with Agent (n=101) or SeQuent Please (n=49). This trial also includes a separate single-arm substudy evaluating the clinical safety and effectiveness of Agent in patients with ISR. The primary endpoint of 6-month target lesion failure (TLF) was observed in 3.0% of Agent and 0.0% of SeQuent Please patients (difference=3.0%; 97.5% upper confidence bound [UCB]=9.57%, which is less than the prespecified margin of 13.2%; Pnon-inferiority=0.0012). There were no deaths or thrombosis, and angiographic and quality-of-life outcomes were comparable between groups. The AGENT Japan ISR substudy (n=30) primary endpoint was met because the one-sided 97.5% UCB for 6-month TLF (3.3%) was significantly less than the study success criterion of 15.1% (97.5% UCB=9.8%; P<0.0001). CONCLUSIONS: Data from this study demonstrate good clinical outcomes with the Agent DCB when used to treat patients with SVD or ISR.

Impact of "black rock" on clinical outcomes after endovascular therapy for de novo calcified femoropopliteal lesions.

The relationship between severity of calcification and clinical outcomes after endovascular therapy (EVT) for femoropopliteal lesions is well known. We often encounter dense calcifications in our daily practice, which are darker than normal calcifications on angiography. Accordingly, we named it "black rock" (BR), and investigated its impact on clinical outcomes after EVT. We retrospectively analyzed 677 lesions in 495 patients who underwent EVT for de novo calcified femoropopliteal lesions at our hospital between April 2007 and June 2020. BR is defined as a calcification which is 1 cm or more in length, occupies more than half of the vessel diameter, and appears darker than the body of the femur on angiography. Propensity score matching analysis was performed to compare clinical outcomes between lesions with BR [BR (+) group] and without BR [BR (-) group]. A total of 119 matched pairs of lesions were analyzed. Primary patency at 2 years was significantly lower in the BR (+) group than in the BR (-) group (48% vs. 75%, p = .0007). Multivariate analysis revealed that the presence of BR [hazard ratio (HR) = 2.23, 95% confidence interval (CI); 1.48-3.38, p = .0001], lesion length (HR = 1.03, 95%CI; 1.00-1.06, p = .0244), and no scaffold use (HR = 1.58, 95%CI; 1.06-2.36, p = .0246) were predictors of restenosis. The presence of BR is independently associated with clinical outcomes after EVT for de novo calcified femoropopliteal lesions.

Effect of Nonsupplemented Low-Protein Diet on the Initiation of Renal Replacement Therapy in Stage 4 and 5 Chronic Kidney Disease: A Retrospective Multicenter Cohort Study in Japan.

OBJECTIVE: In subjects with chronic kidney disease (CKD), the effect of low-protein diet (LPD) is expected to alleviate uremic symptoms. However, whether LPD is effective in preventing loss of kidney function is controversial. The aim of this study was to evaluate the association between LPD and renal outcomes. METHODS: We conducted a multicenter cohort study of 325 patients who suffered CKD stage 4 and 5 with eGFR ≥10 mL/min/1.73 m,2 between January 2008 and December 2014. The primary diseases of the patients were chronic glomerulonephritis (47.7%), nephrosclerosis (16.9%), diabetic nephropathy (26.2%), and others (9.2%). The patients were divided into four groups, based on the mean protein intake (PI)/day, group 1 (n = 76): PI < 0.5 g/kg ideal body weight/day, group 2 (n = 56): 0.5 ≤ PI < 0.6 g/kg/day, group 3 (n = 110): 0.6 ≤ PI < 0.8 g/kg/day, group 4 (n = 83): PI ≥ 0.8 g/kg/day. Dietary supplementation with essential amino acids and ketoanalogues was not used. The outcome measure was occurrence of renal replacement therapy (RRT) (hemodialysis, peritoneal dialysis, renal transplantation (excluding preemptive transplantation)) and all-cause mortality until December 2018. Cox regression models were used to examine whether LPD was associated with the risk of outcomes. RESULTS: During a mean follow-up of 4.1 ± 2.2 years. Thirty-three patients (10.2%) died of all causes, 163 patients (50.2%) needed to start RRT, and 6 patients (1.8%) received a renal transplant. LPD therapy of 0.5 g/kg/day or less was significantly related to a lower risk of RRT and all-cause mortality [Hazard ratio = 0.656; 95% confidence interval, 0.438 to 0.984, P = .042]. CONCLUSIONS: These results suggest that non-supplemented LPD therapy of 0.5 g/kg/day or less may prolong the initiation of RRT in stage 4 and 5 CKD patients.

Intensity Ratio of Kß/Kα in Selected Elements from Mg to Cu, and the Chemical Effects of Cr Kα1,2 Diagram Lines and Cr Kß/Kα Intensity Ratio in Cr Compounds.

Kα,ß X-ray lines from photon excitation were measured in selected elements from Mg to Cu using a high-resolution double-crystal X-ray spectrometer with a proportional counter, and the Kß/Kα intensity ratio for each element was obtained, after correcting for self-absorption, detection efficiency, and crystal reflectance. This intensity ratio increases rapidly from Mg to Ca but, in the 3d elements region, the increase becomes slower. This is related to the intensity of the Kß line involving valence electrons. The slow increase of this ratio in the 3d elements region is thought to be due to the correlation between 3d and 4s electrons. Moreover, the chemical shifts, FWHM, asymmetry indices, and Kß/Kα intensity ratios of the Cr compounds, due to different valences, were also investigated using the same double-crystal X-ray spectrometer. The chemical effects were clearly observed, and the Kß/Kα intensity ratio was found to be compound-dependent for Cr.

Induction of Gastric Cancer by Successive Oncogenic Activation in the Corpus.

BACKGROUND & AIMS: Metaplasia and dysplasia in the corpus are reportedly derived from de-differentiation of chief cells. However, the cellular origin of metaplasia and cancer remained uncertain. Therefore, we investigated whether pepsinogen C (PGC) transcript-expressing cells represent the cellular origin of metaplasia and cancer using a novel Pgc-specific CreERT2 recombinase mouse model. METHODS: We generated a Pgc-mCherry-IRES-CreERT2 (Pgc-CreERT2) knock-in mouse model. Pgc-CreERT2/+ and Rosa-EYFP mice were crossed to generate Pgc-CreERT2/Rosa-EYFP (Pgc-CreERT2/YFP) mice. Gastric tissues were collected, followed by lineage-tracing experiments and histologic and immunofluorescence staining. We further established Pgc-CreERT2;KrasG12D/+ mice and investigated whether PGC transcript-expressing cells are responsible for the precancerous state in gastric glands. To investigate cancer development from PGC transcript-expressing cells with activated Kras, inactivated Apc, and Trp53 signaling pathways, we crossed Pgc-CreERT2/+ mice with conditional KrasG12D, Apcflox, Trp53flox mice. RESULTS: Expectedly, mCherry mainly labeled chief cells in the Pgc-CreERT2 mice. However, mCherry was also detected throughout the neck cell and isthmal stem/progenitor regions, albeit at lower levels. In the Pgc-CreERT2;KrasG12D/+ mice, PGC transcript-expressing cells with KrasG12D/+ mutation presented pseudopyloric metaplasia. The early induction of proliferation at the isthmus may reflect the ability of isthmal progenitors to react rapidly to Pgc-driven KrasG12D/+ oncogenic mutation. Furthermore, Pgc-CreERT2;KrasG12D/+;Apcflox/flox mice presented intramucosal dysplasia/carcinoma and Pgc-CreERT2;KrasG12D/+;Apcflox/flox;Trp53flox/flox mice presented invasive and metastatic gastric carcinoma. CONCLUSIONS: The Pgc-CreERT2 knock-in mouse is an invaluable tool to study the effects of successive oncogenic activation in the mouse corpus. Time-course observations can be made regarding the responses of isthmal and chief cells to oncogenic insults. We can observe stomach-specific tumorigenesis from the beginning to metastatic development.

Procedural results and in-hospital outcomes of percutaneous coronary intervention for chronic total occlusion in patients with reduced left ventricular ejection fraction: Sub-analysis of the Japanese CTO-PCI Expert Registry.

OBJECTIVES: To evaluate the procedural results and in-hospital outcomes of percutaneous coronary intervention (PCI) for chronic total occlusion (CTO) in patients with reduced left ventricular ejection fraction (LVEF). BACKGROUND: While the technical success of general CTO-PCI has improved, CTO-PCI patients with reduced LVEF remain at high-risk for adverse events. METHODS: The data of 820 patients with LVEF ≤ 35% (Group 1), 1816 patients with LVEF = 35%-50% (Group 2), and 5503 patients with LVEF ≥ 50% (Group 3), registered in the Japanese CTO-PCI Expert Registry from January 2014 to December 2019, were retrospectively analyzed. The primary endpoint was in-hospital major adverse cardiac or cerebrovascular events (MACCEs), including death, myocardial infarction, stent thrombosis, stroke, and emergent revascularization. Secondary endpoints included procedural details, guidewire success, and technical success. RESULTS: There were no differences in guidewire and technical success rates between the groups. In-hospital MACCEs was significantly higher in Group 1 (Group 1 vs. Group 2 vs. Group 3: 3.4% vs. 1.7% vs. 1.5%, p = 0.001) and was especially driven by death (1.3% vs. 0.3% vs. 0.1%, p < 0.001) and stroke (0.7% vs. 0.2% vs. 0.2%, p = 0.007). Multivariate analysis showed that LVEF ≤ 35% (odds ratio [OR]; 1.58, 95% confidence interval [CI]; 1.04-2.41, p = 0.03) and New York Heart Association (NYHA) class ≥ 3 (OR; 2.01, 95% CI; 1.03-3.93, p = 0.04) were predictors of in-hospital MACCEs. CONCLUSIONS: In-hospital MACCEs were significantly higher in patients with LVEF ≤ 35%. LVEF ≤;35% and NYHA class ≥ 3 were predictors of in-hospital MACCEs after CTO-PCI.

Predictive performance of J-Calc-CTO score for guidewire crossing through chronic total occlusion lesions within 30 minutes: J-CTO score modified by computed tomography coronary angiography.

The original J-CTO score predicts the difficulty of percutaneous coronary intervention (PCI) for chronic total occlusion (CTO) lesions, but the grade of calcification has not been fully evaluated. We examined 137 patients (141 CTO lesions) who underwent coronary computed tomography angiography (CTA) pre-PCI between October 2016 and October 2021. They were randomly divided into derivation (n = 94) and validation (n = 47) groups. The degree and distribution of calcification in the occluded segment were assessed using CTA. The calcified index was defined as calcium volume divided by the length of the occluded segment. We created the J-Calc-CTO score consisting of calcification parameters associated with 30-min wire crossing in the derivation group. The validity of the J-Calc-CTO score was compared with that of the original J-CTO score using c-statistics. The procedural success rate was 96%, and 30-min wire crossing during the procedure was achieved in 29%. Dense calcification (calcified-index >12) (odds ratio [OR]: 4.63; 95% confidence interval [CI]: 1.24-22.2; p = 0.04) and calcification in the center of the lumen (OR: 7.25; 95% CI: 1.48-32.1; p = 0.02) were independently associated with 30-min wire crossing as variables evaluated using CTA. The J-Calc-CTO score was created by adding 1 point to the two parameters in place of "calcification" in the original J-CTO score. The J-Calc-CTO score showed a higher predictive value of 30-min wire crossing than the J-CTO score in the derivation (c-statistics; 0.836 vs. 0.670; p > 0.01) and validation groups (c-statistics; 0.879 vs. 0.767, p > 0.01). The degree and distribution of calcification evaluated using CTA refined the predictive value of the original J-CTO score for 30-min wire crossing.

Scoring Model to Predict Major Amputation in Patients With Chronic Limb-Threatening Ischemia at Wound, Ischemia, and Foot Infection Clinical Stage 4 After Endovascular Therapy.

PURPOSE: We investigated the predictors of major amputation (MA) at 1 year and prepared a scoring model to stratify the clinical outcomes of chronic limb-threatening ischemia (CLTI) patients at wound, ischemia, and foot infection (WIfI) clinical stage 4 after endovascular therapy (EVT). MATERIALS AND METHODS: This study was a retrospective, observational study performed at a single center. A total of 353 CLTI patients (390 limbs) were treated with EVT between April 2007 and December 2016. Among these, limbs at WIfI clinical stages 1, 2, and 3 were excluded, and 194 limbs at WIfI clinical stage 4 (49.7%) were enrolled. The primary endpoint was major amputation (MA) free rate at 1 year. Predictors of MA at 1 year was evaluated by Cox proportional hazard analysis. RESULTS: At 1 year, the incidence of MA was 18.0% (35 limbs). Cox proportional hazard analysis revealed that hemodialysis (hazard ratio [HR] 2.63; 95% confidence interval [CI], 1.24-5.58; p=0.012), fI3 (HR 2.54; 95% CI, 1.28-5.06; p=0.008), toe wounds (HR 0.29; 95% CI, 0.094-0.88; p=0.029), and visible blood flow to the wound (HR 0.43; 95% CI, 0.21-0.89; p=0.023) were associated with MA. We assigned 1 point for positive predictors of MA, hemodialysis, and fI3; 1 point was deducted for negative predictors of MA, toe wounds, and visible blood flow to the wound. A score of -2 or -1, was defined as the low-risk group, 0 was defined as the intermediate-risk group, and +1 or +2 were defined as the high-risk group. At 1 year, MA free rate, wound healing rate, and amputation-free survival rate were stratified according to a scoring model. MA free rate was 96.6% in low-risk, 72.4% in intermediate-risk, and 67.3% in high-risk (p<0.001); wound healing rate was 67.8% in low-risk, 27.6% in intermediate-risk, and 4.1% in high-risk (p<0.001); amputation-free survival rate was 65.3% in low-risk, 44.8% in intermediate-risk, and 18.4% in high-risk (p<0.001). CONCLUSIONS: The scoring model based on the predictors of MA stratified clinical outcomes in CLTI patients at WIfI clinical stage 4.

Scoring model to predict low image quality of drug-eluting stent evaluated by computed tomography coronary angiography.

Evaluation of in-stent restenosis (ISR) by computed tomography coronary angiography (CTCA) is less invasive but often impossible. We aimed to create a scoring model for predicting which drug-eluting stents (DES) cannot be evaluated with CTCA. We enrolled 757 consecutive implanted DES assessed with CTCA. Non-diagnostic evaluation was defined as poor/not evaluative by two different observers. These stents were randomly divided into a derivation (n = 379) and validation (n = 378) group. In the derivation group, we assessed predictors using logistic regression analysis and created a scoring model that would stratify non-diagnostic evaluation of DES-ISR. The validity of this scoring model was evaluated in the validation group using receiver-operating characteristic analysis. The percentage of non-diagnostic stents was 19/21% in the derivation/validation group (p = 0.71). Non-diagnostic evaluation was independently associated with implanted stent diameter (2.25-2.5. vs. 2.5-3 vs. > 3.0 mm), severe calcification, stent-in-stent lesion, and type of DES (stainless vs. CoCr vs. PtCr) in the derivation group. The predicting system of implanted DES non-diagnostic by CTCA (PIDENT) for non-diagnostic evaluation, including these four baseline factors, was derived (C-statistic = 0.86 in derivation group, cutoff: 8 points). The PIDENT score had a high predictive value for non-diagnostic DES in the validation model (C-statistic = 0.87, sensitivity 86%, specificity 74%, cutoff 8 points, p < 0.001). The PIDENT score, consisting of baseline characteristics including implanted stent diameter, severe calcification, stent-in-stent lesion, and type of DES, could identify non-diagnostic evaluation of DES-ISR with CTCA. The PIDENT score was valuable in reducing nonevaluable and meaningless CTCA for DES-ISR.

The H. pylori CagA Oncoprotein Induces DNA Double Strand Breaks through Fanconi Anemia Pathway Downregulation and Replication Fork Collapse.

The proteins from the Fanconi Anemia (FA) pathway of DNA repair maintain DNA replication fork integrity by preventing the unscheduled degradation of nascent DNA at regions of stalled replication forks. Here, we ask if the bacterial pathogen H. pylori exploits the fork stabilisation machinery to generate double stand breaks (DSBs) and genomic instability. Specifically, we study if the H. pylori virulence factor CagA generates host genomic DSBs through replication fork destabilisation and collapse. An inducible gastric cancer model was used to examine global CagA-dependent transcriptomic and proteomic alterations, using RNA sequencing and SILAC-based mass spectrometry, respectively. The transcriptional alterations were confirmed in gastric cancer cell lines infected with H. pylori. Functional analysis was performed using chromatin fractionation, pulsed-field gel electrophoresis (PFGE), and single molecule DNA replication/repair fiber assays. We found a core set of 31 DNA repair factors including the FA genes FANCI, FANCD2, BRCA1, and BRCA2 that were downregulated following CagA expression. H. pylori infection of gastric cancer cell lines showed downregulation of the aforementioned FA genes in a CagA-dependent manner. Consistent with FA pathway downregulation, chromatin purification studies revealed impaired levels of Rad51 but higher recruitment of the nuclease MRE11 on the chromatin of CagA-expressing cells, suggesting impaired fork protection. In line with the above data, fibre assays revealed higher fork degradation, lower fork speed, daughter strands gap accumulation, and impaired re-start of replication forks in the presence of CagA, indicating compromised genome stability. By downregulating the expression of key DNA repair genes such as FANCI, FANCD2, BRCA1, and BRCA2, H. pylori CagA compromises host replication fork stability and induces DNA DSBs through fork collapse. These data unveil an intriguing example of a bacterial virulence factor that induces genomic instability by interfering with the host replication fork stabilisation machinery.

Iqgap3-Ras axis drives stem cell proliferation in the stomach corpus during homoeostasis and repair.

OBJECTIVE: Tissue stem cells are central regulators of organ homoeostasis. We looked for a protein that is exclusively expressed and functionally involved in stem cell activity in rapidly proliferating isthmus stem cells in the stomach corpus. DESIGN: We uncovered the specific expression of Iqgap3 in proliferating isthmus stem cells through immunofluorescence and in situ hybridisation. We performed lineage tracing and transcriptomic analysis of Iqgap3 +isthmus stem cells with the Iqgap3-2A-tdTomato mouse model. Depletion of Iqgap3 revealed its functional importance in maintenance and proliferation of stem cells. We further studied Iqgap3 expression and the associated gene expression changes during tissue repair after tamoxifen-induced damage. Immunohistochemistry revealed elevated expression of Iqgap3 in proliferating regions of gastric tumours from patient samples. RESULTS: Iqgap3 is a highly specific marker of proliferating isthmus stem cells during homoeostasis. Iqgap3+isthmus stem cells give rise to major cell types of the corpus unit. Iqgap3 expression is essential for the maintenance of stem potential. The Ras pathway is a critical partner of Iqgap3 in promoting strong proliferation in isthmus stem cells. The robust induction of Iqgap3 expression following tissue damage indicates an active role for Iqgap3 in tissue regeneration. CONCLUSION: IQGAP3 is a major regulator of stomach epithelial tissue homoeostasis and repair. The upregulation of IQGAP3 in gastric cancer suggests that IQGAP3 plays an important role in cancer cell proliferation.

Inflammatory and mitogenic signals drive interleukin 23 subunit alpha (IL23A) secretion independent of IL12B in intestinal epithelial cells.

The heterodimeric cytokine interleukin-23 (IL-23 or IL23A/IL12B) is produced by dendritic cells and macrophages and promotes the proinflammatory and regenerative activities of T helper 17 (Th17) and innate lymphoid cells. A recent study has reported that IL-23 is also secreted by lung adenoma cells and generates an inflammatory and immune-suppressed stroma. Here, we observed that proinflammatory tumor necrosis factor (TNF)/NF-κB and mitogen-activated protein kinase (MAPK) signaling strongly induce IL23A expression in intestinal epithelial cells. Moreover, we identified a strong crosstalk between the NF-κB and MAPK/ERK kinase (MEK) pathways, involving the formation of a transcriptional enhancer complex consisting of proto-oncogene c-Jun (c-Jun), RELA proto-oncogene NF-κB subunit (RelA), RUNX family transcription factor 1 (RUNX1), and RUNX3. Collectively, these proteins induced IL23A secretion, confirmed by immunoprecipitation of endogenous IL23A from activated human colorectal cancer (CRC) cell culture supernatants. Interestingly, IL23A was likely secreted in a noncanonical form, as it was not detected by an ELISA specific for heterodimeric IL-23 likely because IL12B expression is absent in CRC cells. Given recent evidence that IL23A promotes tumor formation, we evaluated the efficacy of MAPK/NF-κB inhibitors in attenuating IL23A expression and found that the MEK inhibitor trametinib and BAY 11-7082 (an IKKα/IκB inhibitor) effectively inhibited IL23A in a subset of human CRC lines with mutant KRAS or BRAFV600E mutations. Together, these results indicate that proinflammatory and mitogenic signals dynamically regulate IL23A in epithelial cells. They further reveal its secretion in a noncanonical form independent of IL12B and that small-molecule inhibitors can attenuate IL23A secretion.

Efficacy of the novel technique HIRANODOME in preventing distal embolization during endovascular treatment of femoropopliteal lesions.

OBJECTIVES: The efficacy and clinical outcomes of a novel technique "HIRANODOME" (Interim hemostatic technique with HIgh pressure for Regional blood flow in the superficial femoral Artery, NOninvasive Distal protection Occlusion MEthod) in preventing distal embolization during endovascular treatment (EVT) of femoropopliteal lesions were evaluated. BACKGROUND: Distal embolization of femoropopliteal lesions may worsen limb ischemia or cause limb loss. Conventional filter wires are cumbersome and expensive and may cause vessel injury. HIRANODOME can, therefore, be a feasible and noninvasive alternative. METHODS: Between April 2007 and August 2018, 94 consecutive patients who underwent EVT for femoropopliteal lesions along with anticipated distal embolization were identified. About 9 out of these 94 patients were excluded due to filter device use and 8 were excluded due to acute limb ischemia. Consequently, 77 patients using HIRANODOME for distal protection were included. HIRANODOME involved wrapping the Tometakun around the knee to enable external compression of the popliteal artery, thereby blocking the blood flow. The evaluation items were 30-day mortality, 30-day major amputation, prevention from distal embolization, and thrombus capture rate. RESULTS: The mean age was 74.4 ± 10.3 years, 53.2% were men, 55.8% were diabetic, and 9.1% were on hemodialysis. The mean lesion length was 199.5 ± 94.4 mm; 79.2% were class C/D lesions (TASC II classification). The 30-day mortality and major amputation rates were 1.9 and 1.0%, respectively. The rate of prevention of distal embolization or no-flow/slow-flow phenomenon was 93.4%. Capture of thrombus was observed in 66 lesions (62.3%). CONCLUSIONS: The HIRANODOME technique was effective in preventing distal embolization during EVT of femoropopliteal lesions.

Stentless Strategy by Drug-Coated Balloon Angioplasty following Directional Coronary Atherectomy for Left Main Bifurcation Lesion.

AIMS: We aimed to evaluate the efficacy of stentless strategy by drug-coated balloon (DCB) angioplasty following directional coronary atherectomy (DCA) for left main (LM) bifurcation lesions. METHODS: A total of 38 patients who underwent DCB angioplasty following DCA for LM bifurcation lesions were retrospectively enrolled. The primary endpoint was target vessel failure (TVF) at 12 months. Secondary endpoints included procedure-related major events during the hospitalization, major adverse cardiac events at 12 months, ischemia-driven target lesion revascularization (TLR) at 12 months, and bleeding complications defined as the Bleeding Academic Research Consortium criteria ≥2 at 12 months. RESULTS: Among these 38 lesions, 31 lesions were de novo LM bifurcation lesions and 7 lesions were stent edge restenosis at the left anterior descending (LAD) ostium. The mean % plaque area (%PA) after DCA was 44.0 ± 7.4%. TVF at 12 months occurred in 1 lesion (3.2%) of de novo LM bifurcation lesion and in 3 lesions (42.9%) of stent edge restenosis at the LAD ostium. All events of TVF were ischemia-driven TLR by percutaneous coronary intervention. Among 4 TLR cases, %PA after DCA was high (55.9%) in the de novo LM bifurcation lesions; on the other hand, %PA after DCA was low (42.4%, 38.7%, and 25.7% in the 3 cases) in stent edge restenosis at the LAD ostium. No procedure-related major events were observed during hospitalization. There was no cardiac death, no myocardial infarction, no coronary artery bypass grafting, and no bleeding complications at 12 months. CONCLUSIONS: Stentless strategy by DCB angioplasty following DCA for de novo LM bifurcation lesions resulted in acceptable outcomes. On the other hand, its efficacy was limited for stent edge restenosis at the LAD ostium even after aggressive debulking by DCA.