CHK1 attenuates cardiac dysfunction via suppressing SIRT1-ubiquitination.

BACKGROUND: Mitochondrial dysfunction is linked to myocardial ischemia-reperfusion (I/R) injury. Checkpoint kinase 1 (CHK1) could facilitate cardiomyocyte proliferation, however, its role on mitochondrial function in I/R injury remains unknown. METHODS: To investigate the role of CHK1 on mitochondrial function following I/R injury, cardiomyocyte-specific knockout/overexpression mouse models were generated. Adult mouse cardiomyocytes (AMCMs) were isolated for in vitro study. Mass spectrometry-proteomics analysis and protein co-immunoprecipitation assays were conducted to dissect the molecular mechanism. RESULTS: CHK1 was downregulated in myocardium post I/R and AMCMs post oxygen-glucose deprivation/re­oxygenation (OGD/R). In vivo, CHK1 overexpression protected against I/R induced cardiac dysfunction, while heterogenous CHK1 knockout exacerbated cardiomyopathy. In vitro, CHK1 inhibited OGD/R-induced cardiomyocyte apoptosis and bolstered cardiomyocyte survival. Mechanistically, CHK1 attenuated oxidative stress and preserved mitochondrial metabolism in cardiomyocytes under I/R. Moreover, disrupted mitochondrial homeostasis in I/R myocardium was restored by CHK1 through the promotion of mitochondrial biogenesis and mitophagy. Through mass spectrometry analysis following co-immunoprecipitation, SIRT1 was identified as a direct target of CHK1. The 266-390 domain of CHK1 interacted with the 160-583 domain of SIRT1. Importantly, CHK1 phosphorylated SIRT1 at Thr530 residue, thereby inhibiting SMURF2-mediated degradation of SIRT1. The role of CHK1 in maintaining mitochondrial dynamics control and myocardial protection is abolished by SIRT1 inhibition, while inactivated mutation of SIRT1 Thr530 fails to reverse the impaired mitochondrial dynamics following CHK1 knockdown. CHK1 Δ390 amino acids (aa) mutant functioned similarly to full-length CHK1 in scavenging ROS and maintaining mitochondrial dynamics. Consistently, cardiac-specific SIRT1 knockdown attenuated the protective role of CHK1 in I/R injury. CONCLUSIONS: Our findings revealed that CHK1 mitigates I/R injury and restores mitochondrial dynamics in cardiomyocytes through a SIRT1-dependent mechanism.

Ankrd1 regulates endogenous cardiac regeneration in mice by modulating cyclin D1.

Restoration of the expression of factors regulating neonatal heart regeneration in the adult heart can promote myocardial repair. Therefore, investigations of the regulatory factors that play key roles in neonatal heart regeneration are urgently needed for the development of cardiac regenerative therapies. In our previous study, we identified ankyrin repeat domain 1 (Ankrd1) through multiomics analysis in a neonatal mouse model of cardiac regeneration and hypothesized that Ankrd1 plays a regulatory role in neonatal heart regeneration. In the present study, we aimed to determine the role of Ankrd1 in neonatal heart regeneration and adult myocardial repair. Our findings confirmed that Ankrd1 could mediate cardiomyocyte proliferation and that Ankrd1 knockdown in cardiomyocytes inhibited myocardial regeneration after apical resection in neonatal mice. Furthermore, we found that cardiomyocyte-specific Ankrd1 overexpression promoted cardiac repair and cardiac function recovery after adult myocardial infarction (MI). Mechanistically, Ankrd1 could regulate the cell cycle of cardiomyocytes and significantly mediate cardiac regeneration, at least in part, through cyclin D1. Overall, our study demonstrates that Ankrd1 is an effective target for achieving cardiac repair after MI, providing new ideas for the treatment of ischemic heart disease in the future.

Circular RNA IGF1R Promotes Cardiac Repair via Activating ß-Catenin Signaling by Interacting with DDX5 in Mice after Ischemic Insults.

The potential of circular RNAs (circRNAs) as biomarkers and therapeutic targets is becoming increasingly evident, yet their roles in cardiac regeneration and myocardial renewal remain largely unexplored. Here, we investigated the function of circIGF1R and related mechanisms in cardiac regeneration. Through analysis of circRNA sequencing data from neonatal and adult cardiomyocytes, circRNAs associated with regeneration were identified. Our data showed that circIGF1R expression was high in neonatal hearts, decreased with postnatal maturation, and up-regulated after cardiac injury. The elevation was validated in patients diagnosed with acute myocardial infarction (MI) within 1 week. In human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and myocardial tissue from mice after apical resection and MI, we observed that circIGF1R overexpression enhanced cardiomyocyte proliferation, reduced apoptosis, and mitigated cardiac dysfunction and fibrosis, while circIGF1R knockdown impeded endogenous cardiac renewal. Mechanistically, we identified circIGF1R binding proteins through circRNA precipitation followed by mass spectrometry. RNA pull-down Western blot and RNA immunoprecipitation demonstrated that circIGF1R directly interacted with DDX5 and augmented its protein level by suppressing ubiquitin-dependent degradation. This subsequently triggered the ß-catenin signaling pathway, leading to the transcriptional activation of cyclin D1 and c-Myc. The roles of circIGF1R and DDX5 in cardiac regeneration were further substantiated through site-directed mutagenesis and rescue experiments. In conclusion, our study highlights the pivotal role of circIGF1R in facilitating heart regeneration and repair after ischemic insults. The circIGF1R/DDX5/ß-catenin axis emerges as a novel therapeutic target for enhancing myocardial repair after MI, offering promising avenues for the development of regenerative therapies.

PEX3 promotes regenerative repair after myocardial injury in mice through facilitating plasma membrane localization of ITGB3.

The peroxisome is a versatile organelle that performs diverse metabolic functions. PEX3, a critical regulator of the peroxisome, participates in various biological processes associated with the peroxisome. Whether PEX3 is involved in peroxisome-related redox homeostasis and myocardial regenerative repair remains elusive. We investigate that cardiomyocyte-specific PEX3 knockout (Pex3-KO) results in an imbalance of redox homeostasis and disrupts the endogenous proliferation/development at different times and spatial locations. Using Pex3-KO mice and myocardium-targeted intervention approaches, the effects of PEX3 on myocardial regenerative repair during both physiological and pathological stages are explored. Mechanistically, lipid metabolomics reveals that PEX3 promotes myocardial regenerative repair by affecting plasmalogen metabolism. Further, we find that PEX3-regulated plasmalogen activates the AKT/GSK3ß signaling pathway via the plasma membrane localization of ITGB3. Our study indicates that PEX3 may represent a novel therapeutic target for myocardial regenerative repair following injury.

Checkpoint Kinase 1 Stimulates Endogenous Cardiomyocyte Renewal and Cardiac Repair by Binding to Pyruvate Kinase Isoform M2 C-Domain and Activating Cardiac Metabolic Reprogramming in a Porcine Model of Myocardial Ischemia/Reperfusion Injury.

BACKGROUND: The regenerative capacity of the adult mammalian hearts is limited. Numerous studies have explored mechanisms of adult cardiomyocyte cell-cycle withdrawal. This translational study evaluated the effects and underlying mechanism of rhCHK1 (recombinant human checkpoint kinase 1) on the survival and proliferation of cardiomyocyte and myocardial repair after ischemia/reperfusion injury in swine. METHODS AND RESULTS: Intramyocardial injection of rhCHK1 protein (1 mg/kg) encapsulated in hydrogel stimulated cardiomyocyte proliferation and reduced cardiac inflammation response at 3 days after ischemia/reperfusion injury, improved cardiac function and attenuated ventricular remodeling, and reduced the infarct area at 28 days after ischemia/reperfusion injury. Mechanistically, multiomics sequencing analysis demonstrated enrichment of glycolysis and mTOR (mammalian target of rapamycin) pathways after rhCHK1 treatment. Co-Immunoprecipitation (Co-IP) experiments and protein docking prediction showed that CHK1 (checkpoint kinase 1) directly bound to and activated the Serine 37 (S37) and Tyrosine 105 (Y105) sites of PKM2 (pyruvate kinase isoform M2) to promote metabolic reprogramming. We further constructed plasmids that knocked out different CHK1 and PKM2 amino acid domains and transfected them into Human Embryonic Kidney 293T (HEK293T) cells for CO-IP experiments. Results showed that the 1-265 domain of CHK1 directly binds to the 157-400 amino acids of PKM2. Furthermore, hiPSC-CM (human iPS cell-derived cardiomyocyte) in vitro and in vivo experiments both demonstrated that CHK1 stimulated cardiomyocytes renewal and cardiac repair by activating PKM2 C-domain-mediated cardiac metabolic reprogramming. CONCLUSIONS: This study demonstrates that the 1-265 amino acid domain of CHK1 binds to the 157-400 domain of PKM2 and activates PKM2-mediated metabolic reprogramming to promote cardiomyocyte proliferation and myocardial repair after ischemia/reperfusion injury in adult pigs.

Neutrophil-derived nanovesicles deliver IL-37 to mitigate renal ischemia-reperfusion injury via endothelial cell targeting.

Renal ischemia-reperfusion injury (IRI) is one of the most important causes of acute kidney injury (AKI). Interleukin (IL)-37 has been suggested as a novel anti-inflammatory factor for the treatment of IRI, but its application is still limited by its low stability and delivery efficiency. In this study, we reported a novel engineered method to efficiently and easily prepare neutrophil membrane-derived vesicles (N-MVs), which could be utilized as a promising vehicle to deliver IL-37 and avoid the potential side effects of neutrophil-derived natural extracellular vesicles. N-MVs could enhance the stability of IL-37 and targetedly deliver IL-37 to damaged endothelial cells of IRI kidneys via P-selectin glycoprotein ligand-1 (PSGL-1). In vitro and in vivo evidence revealed that N-MVs encapsulated with IL-37 (N-MV@IL-37) could inhibit endothelial cell apoptosis, promote endothelial cell proliferation and angiogenesis, and decrease inflammatory factor production and leukocyte infiltration, thereby ameliorating renal IRI. Our study establishes a promising delivery vehicle for the treatment of renal IRI and other endothelial damage-related diseases.

Cell sheet formation enhances the therapeutic effects of adipose-derived stromal vascular fraction on urethral stricture.

Urethral stricture (US) is a common disease in urology, lacking effective treatment options. Although injecting a stem cells suspension into the affected area has shown therapeutic benefits, challenges such as low retention rate and limited efficacy hinder the clinical application of stem cells. This study evaluates the therapeutic impact and the mechanism of adipose-derived vascular fraction (SVF) combined with cell sheet engineering technique on urethral fibrosis in a rat model of US. The results showed that SVF-cell sheets exhibit positive expression of α-SMA, CD31, CD34, Stro-1, and eNOS. In vivo study showed less collagen deposition, low urethral fibrosis, and minimal tissue alteration in the group receiving cell sheet transplantation. Furthermore, the formation of a three-dimensional (3D) tissue-like structure by the cell sheets enhances the paracrine effect of SVF, facilitates the infiltration of M2 macrophages, and suppresses the TGF-ß/Smad2 pathway through HGF secretion, thereby exerting antifibrotic effects. Small animal in vivo imaging demonstrates improved retention of SVF cells at the damaged urethra site with cell sheet application. Our results suggest that SVF combined with cell sheet technology more efficiently inhibits the early stages of urethral fibrosis.

Autologous Endothelial Progenitor Cells and Bioactive Factors Improve Bladder Regeneration.

Insufficient vascularization is still a challenge that impedes bladder tissue engineering and results in unsatisfied smooth muscle regeneration. Since bladder regeneration is a complex articulated process, the aim of this study is to investigate whether combining multiple pathways by exploiting a combination of biomaterials, cells, and bioactive factors, contributes to the improvements of smooth muscle regeneration and vascularization in tissue-engineered bladder. Autologous endothelial progenitor cells (EPCs) and bladder smooth muscle cells (BSMCs) are cultured and incorporated into our previously prepared porcine bladder acellular matrix (BAM) for bladder augmentation in rabbits. Simultaneously, exogenous vascular endothelial growth factor (VEGF) and platelet-derived growth factor BB (PDGF-BB) mixed with Matrigel were injected around the implanted cells-BAM complex. In the results, compared with control rabbits received bladder augmentation with porcine BAM seeded with BSMCs, the experimental animals showed significantly improved smooth muscle regeneration and vascularization, along with more excellent functional recovery of tissue-engineered bladder, due to the additional combination of autologous EPCs and bioactive factors, including VEGF and PDGF-BB. Furthermore, cell tracking suggested that the seeded EPCs could be directly involved in neovascularization. Therefore, it may be an effective method to combine multiple pathways for tissue-engineering urinary bladder.

Negatively charged bladder acellular matrix loaded with positively charged adipose-derived mesenchymal stem cell-derived small extracellular vesicles for bladder tissue engineering.

Adipose-derived mesenchymal stem cell-derived small extracellular vesicles (Ad-MSC-sEVs/AMEs) combined with scaffold materials are used in tissue-engineered bladders; however, the lack of retention leads to limited distribution of AMEs in the scaffold areas and low bioavailability of AMEs after bladder reconstruction. To improve retention of AMEs, we developed a novel strategy that modifies the surface charge of the bladder acellular matrix (BAM) via oxidative self-polymerization of dopamine-reducing graphene oxide (GO) and AMEs using ε-polylysine-polyethylene-distearyl phosphatidylethanolamine (PPD). We evaluated two BAM surface modification methods and evaluated the biocompatibility of materials and PPD and electrostatic adherence effects between PPD-modified AMEs and rGO-PDA/BAM in vivo and in vitro. Surface modification increased retention of AMEs, enhanced regeneration of bladder structures, and increased electrical conductivity of rGO-PDA/BAM, thereby improving bladder function recovery. RNA-sequencing revealed 543 miRNAs in human AMEs and 514 miRNAs in rat AMEs. A Venn diagram was used to show target genes of miRNA with the highest proportion predicted by the four databases; related biological processes and pathways were predicted by KEGG and GO analyses. We report a strategy for improving bioavailability of AMEs for bladder reconstruction and reveal that enriched miR-21-5p targets PIK3R1 and activates the PI3K/Akt pathway to promote cell proliferation and migration.

Porous gelatin microspheres implanted with adipose mesenchymal stromal cells promote angiogenesis via protein kinase B/endothelial nitric oxide synthase signaling pathway in bladder reconstruction.

BACKGROUND AIMS: Cell failure and angiogenesis are the key to bladder wall regeneration. Three-dimensional (3D) culture using porous gelatin microspheres (GMs) as a vehicle promotes stem cell proliferation and improves the paracrine capacity of cells. This study aimed to evaluate the therapeutic potential of GMs constructed from adipose-derived mesenchymal stromal cells (ADSCs) (ADSC-GMs) combined with bladder acellular matrix (BAM) in tissue-engineered bladders. METHODS: Isolation of ADSCs, flow cytometry, scanning electron microscopy and cell counting kit-8, ß-galactosidase and enzyme-linked immunosorbent assays were performed in vitro to compare two-dimensional (2D) and 3D cultures. In the in vivo study, male Sprague-Dawley rats were randomly divided into three groups: the BAM replacement alone (BAM) group, ADSCs grown on BAM in replacement (ADSC) group and ADSC-GMs combined with BAM followed by replacement (ADSC-GM) group. Bladder function assessed by urodynamics after 12 weeks of bladder replacement, and the rats were sacrificed at 4 and 12 weeks for further experiments. RESULTS: The in vitro results showed that GM culture promoted ADSC proliferation, inhibited apoptosis and delayed senescence compared with those in the 2D culture. In addition, ADSC-GMs increased the secretion of the angiogenic factors vascular endothelial growth factor, platelet-derived growth factor-BB, and basal fibroblast growth factor. In vivo experiments revealed that ADSC-GMs adhered to the BAM for longer than ADSCs. Moreover, ADSC-GMs significantly promoted the regeneration of bladder vessels and smooth muscle, thereby facilitating the recovery of bladder function. The expression of phosphorylated protein kinase B (AKT) and phosphorylated endothelial nitric oxide synthase (eNOS) was significantly greater in the ADSC-GMs group compared with the BAM and ADSCs groups. CONCLUSIONS: ADSC-GMs increased retention of ADSCs on the BAM, thereby promoting the regeneration and functional recovery of the bladder tissue. ADSC-GMs promoted angiogenesis by activating the AKT/eNOS pathway.

SIRT3-dependent mitochondrial redox homeostasis mitigates CHK1 inhibition combined with gemcitabine treatment induced cardiotoxicity in hiPSC-CMs and mice.

Administration of CHK1-targeted anticancer therapies is associated with an increased cumulative risk of cardiac complications, which is further amplified when combined with gemcitabine. However, the underlying mechanisms remain elusive. In this study, we generated hiPSC-CMs and murine models to elucidate the mechanisms underlying CHK1 inhibition combined with gemcitabine-induced cardiotoxicity and identify potential targets for cardioprotection. Mice were intraperitoneally injected with 25 mg/kg CHK1 inhibitor AZD7762 and 20 mg/kg gemcitabine for 3 weeks. hiPSC-CMs and NMCMs were incubated with 0.5 uM AZD7762 and 0.1 uM gemcitabine for 24 h. Both pharmacological inhibition or genetic deletion of CHK1 and administration of gemcitabine induced mtROS overproduction and pyroptosis in cardiomyocytes by disrupting mitochondrial respiration, ultimately causing heart atrophy and cardiac dysfunction in mice. These toxic effects were further exacerbated with combination administration. Using mitochondria-targeting sequence-directed vectors to overexpress CHK1 in cardiomyocyte (CM) mitochondria, we identified the localization of CHK1 in CM mitochondria and its crucial role in maintaining mitochondrial redox homeostasis for the first time. Mitochondrial CHK1 function loss mediated the cardiotoxicity induced by AZD7762 and CHK1-knockout. Mechanistically, mitochondrial CHK1 directly phosphorylates SIRT3 and promotes its expression within mitochondria. On the contrary, both AZD7762 or CHK1-knockout and gemcitabine decreased mitochondrial SIRT3 abundance, thus resulting in respiration dysfunction. Further hiPSC-CMs and mice experiments demonstrated that SIRT3 overexpression maintained mitochondrial function while alleviating CM pyroptosis, and thereby improving mice cardiac function. In summary, our results suggest that targeting SIRT3 could represent a novel therapeutic approach for clinical prevention and treatment of cardiotoxicity induced by CHK1 inhibition and gemcitabine.

Construction of tissue-engineered bladders using an artificial acellular nanocomposite scaffold loaded with stromal vascular fraction secretome.

Tissue engineering approaches offer promising alternative strategies for reconstructing bladder tissue; however, the low retention of transplanted cells and the possible risk of rejection limit their therapeutic efficacy. Clinical applicability is further limited by the lack of suitable scaffold materials to support the needs of various cell types. In the present study, we developed an artificial nanoscaffold system consisting of stromal vascular fraction (SVF) secretome (Sec) loaded onto zeolitic imidazolate framework-8 (ZIF-8) nanoparticles, which were then incorporated into bladder acellular matrix. This artificial acellular nanocomposite scaffold (ANS) can achieve gradient degradation and slowly release SVF-Sec to promote tissue regeneration. Furthermore, even after long-term cryopreservation, this completely acellular bladder nanoscaffold material still maintains its efficacy. In a rat bladder replacement model, ANS transplantation demonstrated potent proangiogenic ability and induced M2 macrophage polarization to promote tissue regeneration and restore bladder function. Our study demonstrates the safety and efficacy of the ANS, which can play a stem cell-like role while avoiding the disadvantages of cell therapy. Furthermore, the ANS can replace the bladder regeneration model based on cell-binding scaffold materials and has the potential for clinical application. STATEMENT OF SIGNIFICANCE: This study aimed to develop a gradient-degradable artificial acellular nanocomposite scaffold (ANS) loaded with stromal vascular fraction (SVF) secretome for rehabilitating bladders. Using various in vitro methods as well as rat- and zebrafish-based in vivo models, the developed ANS was assessed for efficacy and safety. Results indicated that the ANS achieved gradient degradation and slowly released the SVF secretome to promote tissue regeneration, even after long-term cryopreservation. Furthermore, ANS transplantation demonstrated a potent pro-angiogenic ability and induced M2 macrophage polarization to promote tissue regeneration and restore bladder function in a bladder replacement model. Our study demonstrates that ANS may replace bladder regeneration models based on cell-binding scaffold materials and have potential clinical application.

Association between papillary thyroid carcinoma and cervical lymph node metastasis based on ultrasonic radio frequency signals.

OBJECTIVE: Papillary thyroid carcinoma (PTC) has a high propensity for cervical lymph node metastasis (CLNM). We evaluated the association between PTC radio frequency (RF) signals and CLNM. METHODS: Patients with PTC (n = 170) confirmed by pathology after thyroidectomy between July 2019 and May 2022 were enrolled in this retrospective cohort study. Patients were divided into positive and negative groups according to CLNM. Univariate analysis was performed to predict CLNM and a receiver operating characteristic (ROC) curve was generated to evaluate the diagnostic performance of RF signals and the Thyroid imaging Reporting and Data System. RESULTS: Of 170 patients with 182 nodules included in the study, 11 had multiple nodules. Univariate analysis showed that age, maximum tumor diameter, cross-sectional and longitudinal aspect ratio, RF quantitative parameters (cross-sectional intercept, mid-band, S1, and S4, and longitudinal Higuchi, slope, intercept, mid-band, S1), and echogenic foci were independently associated with CLNM (p < 0.05). The area under the curve (AUC) values of the maximum tumor diameter, longitudinal slope, and echogenic foci were 0.68, 0.61, and 0.62, respectively. Linear regression analysis of maximum tumor diameter, longitudinal slope, and echogenic foci showed that the correlations between longitudinal slope and CLNM were greater than that of echogenic foci (ß = 0.203 vs. ß = 0.154). CONCLUSION: Longitudinal slope and echogenic foci have similar diagnostic efficacy for predicting the risk of CLNM in PTC, although longitudinal slope has a greater correlation with CLNM.

Immunomagnetic Delivery of Adipose-Derived Endothelial Progenitor Cells for the Repair of Renal Ischemia-Reperfusion Injury in a Rat Model.

Renal ischemia-reperfusion injury (IRI) is a significant cause of acute kidney injury (AKI) and usually brings severe public health consequences. Adipose-derived endothelial progenitor cell (AdEPCs) transplantation is beneficial for AKI but suffers from low delivery efficiency. This study was conducted to explore the protective effects of magnetically delivered AdEPCs on the repair of renal IRI. Two types of magnetic delivery methods, namely the endocytosis magnetization (EM) method and the immunomagnetic (IM) method were fabricated using PEG@Fe3O4 and CD133@Fe3O4, and their cytotoxicities in AdEPCs were assessed. In the renal IRI rat model, magnetic AdEPCs were injected via the tail vein and a magnet was placed beside the injured kidney for magnetic guidance. The distribution of transplanted AdEPCs, renal function, and tubular damage were evaluated. Our results suggested that CD133@Fe3O4 had the minimum negative effects on the proliferation, apoptosis, angiogenesis, and migration of AdEPCs compared with PEG@Fe3O4. Renal magnetic guidance could significantly enhance the transplantation efficiency and the therapeutic outcomes of AdEPCs-PEG@Fe3O4 and AdEPCs-CD133@Fe3O4 in the injured kidneys. However, under renal magnetic guidance, AdEPCs-CD133@Fe3O4 had stronger therapeutic effects than PEG@Fe3O4 after renal IRI. The immunomagnetic delivery of AdEPCs with CD133@Fe3O4 could be a promising therapeutic strategy for renal IRI.

Sig1R activates extracellular matrix-induced bladder cancer cell proliferation and angiogenesis by combing ß-integrin.

The extracellular matrix (ECM) regulates many biological functions involved in tumorigenesis and tumor development; however, the underlying mechanism remains unknown. Sigma 1 receptor (Sig1R), a stress-activated chaperone, regulates the crosstalk between the ECM and tumor cells and is related to the malignant characteristics of several tumors. However, the link between Sig1R overexpression and ECM during malignancy has not been established in bladder cancer (BC). Here, we analyzed the interaction of Sig1R and ß-integrin in BC cells and its role in ECM-mediated cell proliferation and angiogenesis. We found that Sig1R forms a complex with ß-integrin to promote ECM-mediated BC cell proliferation and angiogenesis, which enhances the aggressiveness of the tumor cells. This leads to poor survival. Our research revealed that Sig1R mediates the cross-talk between BC cells and their ECM microenvironment, thereby driving the progression of BC. Promisingly, targeting an ion channel function through Sig1R inhibition may serve as a potential approach for BC treatment.

Improved methodology for efficient establishment of the myocardial ischemia-reperfusion model in pigs through the median thoracic incision.

To investigate the feasibility and effectiveness of establishing porcine ischemia-reperfusion models by ligating the left anterior descending (LAD) coronary artery, we first randomly divided 16 male Bama pigs into a sham group and a model group. After anesthesia, we separated the arteries and veins. Subsequently, we rapidly located the LAD coronary artery at the beginning of its first diagonal branch through a mid-chest incision. Then, we loosened and released the ligation line after five minutes of pre-occlusion. Finally, we ligated the LAD coronary artery in situ two minutes later and loosened the ligature 60 min after ischemia. Compared with the sham group, electrocardiogram showed multiple continuous lead ST-segment elevations, and ultrasound cardiogram showed significantly lower ejection fraction and left ventricular fractional shortening at one hour and seven days post-operation in the model group. Twenty-four hours after the operation, cardiac troponin T and creatine kinase-MB isoenzyme levels significantly increased in the model group, compared with the sham group. Hematoxylin and eosin staining showed the presence of many inflammatory cells infiltrating the interstitium of the myocardium in the model group but not in the sham group. Masson staining revealed a significant increase in infarct size in the ischemia/reperfusion group. All eight pigs in the model group recovered with normal sinus heart rates, and the survival rate was 100%. In conclusion, the method can provide an accurate and stable large animal model for preclinical research on ischemia/reperfusion with a high success rate and homogeneity of the myocardial infarction area.

P16INK4a Regulates ROS-Related Autophagy and CDK4/6-Mediated Proliferation: A New Target of Myocardial Regeneration Therapy.

Neonatal mice achieve complete cardiac repair through endogenous myocardial regeneration after apical resection (AR), but this capacity is rapidly lost 7 days after birth. As an upstream inhibitor of cyclin-dependent kinase 4/6- (CDK4/6-) mediated cell cycle activity, p16INK4a is widely involved in regulating tumor and senescence. Given that p16INK4a had a significant negative regulation on cell proliferation, targeting cardiomyocytes (CMs) to inhibit p16INK4a seems to be a promising attempt at myocardial regeneration therapy. The p16INK4a expression was upregulated during perimyocardial regeneration time. Knockdown of p16INK4a stimulated CM proliferation, while p16INK4a overexpression had the opposite effect. In addition, p16INK4a knockdown prolonged the proliferation time window of newborn myocardium. And p16INK4a overexpression inhibited cell cycle activity and deteriorated myocardial regeneration after AR. The quantitative proteomic analysis showed that p16INK4a knockdown mediated the cell cycle progression and intervened in energy metabolism homeostasis. Mechanistically, overexpression of p16INK4a causes abnormal accumulation of reactive oxygen species (ROS) to induce autophagy, while scavenging ROS with N-acetylcysteine can alleviate autophagy and regulate p16INK4a, CDK4/6, and CyclinD1 in a covering manner. And the effect of inhibiting the proliferation of p16INK4a-activated CMs was significantly blocked by the CDK4/6 inhibitor Palbociclib. In summary, p16INK4a regulated CM proliferation progression through CDK4/6 and ROS-related autophagy to jointly affect myocardial regeneration repair. Our study revealed that p16INK4a might be a potential therapeutic target for myocardial regeneration after injury.

A multi-institutional study of association of sonographic characteristics with cervical lymph node metastasis in unifocal papillary thyroid carcinoma.

Objective: Papillary thyroid carcinoma (PTC) is the most common pathological type of thyroid carcinoma, and is prone to cervical lymph node metastases (CLNM). We aim to evaluate the association between sonographic characteristics of PTC and CLNM before the initial surgery. Methods: Clinical information as well as ultrasonographic measurements and characteristics for 2376 patients from three hospitals were acquired in this retrospective cohort study. Univariate and multivariate logistic analysis were performed to predict CLNM in unifocal PTC patients. Receiver operating characteristic (ROC) curve was created to evaluate diagnostic performance. Results: Univariate analysis showed that gender, age, maximum tumor diameter and volume, cross-sectional and longitudinal aspect ratio, location, echogenicity, margin, and echogenic foci were independently associated with CLNM metastatic status (P < 0.05). Multivariate logistic analysis showed that gender, age, maximum tumor diameter and volume, cross-sectional aspect ratio (CSAR), location, echogenicity, margin, and echogenic foci were independent correlative factors; CSAR showed a significant difference for PTC2 to predict CLNM. The area under the curve (AUC) of the maximum tumor diameter, tumor volume, margin, and echogenic foci was 0.70, 0.69, 0.65, and 0.70, respectively. The multiple-variable linear regression model was constructed with an AUC of 0.77, a specificity of 73.4%, and a sensitivity of 72.3%. Kruskal-Wallis analysis for positive subgroups, maximum tumor diameter and volume, cross-sectional and longitudinal aspect ratio, margin, and echogenic foci showed statistical significance (P < 0.05). Conclusions: Younger age (< 55 years), male, larger tumor, and echogenic foci were high risk factors for CLNM in patients with unifocal PTC. CSAR had a more effective predictive value for CLNM in patients with larger thyroid tumors. A larger tumor with irregular and punctate echogenic foci was also more prone to the lateral neck, and both central and lateral neck metastasis.

CDK9 binds and activates SGK3 to promote cardiac repair after injury via the GSK-3ß/ß-catenin pathway.

The mammalian heart possesses entire regeneration capacity after birth, which is lost in adulthood. The role of the kinase network in myocardial regeneration remains largely elusive. SGK3 (threonine-protein kinase 3) is a functional kinase we identified previously with the capacity to promote cardiomyocyte proliferation and cardiac repair after myocardial infarction. However, the upstream signals regulating SGK3 are still unknown. Based on the quantitative phosphoproteomics data and pulldown assay, we identified cyclin-dependent kinase 9 (CDK9) as a novel therapeutic target in regeneration therapy. The direct combination between CDK9 and SGK3 was further confirmed by co-immunoprecipitation (Co-IP). CDK9 is highly expressed in the newborn period and rarely detected in the adult myocardium. In vitro, the proliferation ratio of primary cardiomyocytes was significantly elevated by CDK9 overexpression while inhibited by CDK9 knockdown. In vivo, inhibition of CDK9 shortened the time window of cardiac regeneration after apical resection (AR) in neonatal mice, while overexpression of CDK9 significantly promoted mature cardiomyocytes (CMs) to re-enter the cell cycle and cardiac repair after myocardial infarction (MI) in adult mice. Mechanistically, CDK9 promoted cardiac repair by directly activating SGK3 and downstream GSK-3ß/ß-catenin pathway. Consequently, our study indicated that CDK9 might be a novel target for MI therapy by stimulating myocardial regeneration.

The Role of P4HA1 in Multiple Cancer Types and its Potential as a Target in Renal Cell Carcinoma.

Background: Prolyl 4-hydroxylase subunit alpha 1 (P4HA1) provides the majority of the catalytic site of the active P4H enzyme. Emerging evidence has revealed that P4HA1 participates in the initiation and development of several malignant tumors. However, a pan-cancer analysis of P4HA1 has not been performed. Methods: In this study, we carried out an in-depth analysis of the expression patterns and prognostic value of P4HA1 using the datasets of The Cancer Genome Atlas (TCGA) and Kaplan-Meier Plotter. Genomic and epigenetic alterations of P4HA1 and the correlation of P4HA1 with DNA methylation in different cancers were also analyzed across multiple databases. In addition, the purity-adjusted partial Spearman's correlation test was utilized to evaluate the correlation between P4HA1 expression and immune cell infiltration. We also further explored the biological function and mechanism of P4HA1 in renal cell carcinoma (RCC). Results: We characterized the expression profiles and prognostic values of P4HA1 in multiple cancer types. P4HA1 expression was increased in clear cell renal cell carcinoma (RCC) compared to adjacent normal tissues, and P4HA1 positively correlated with the overall survival (OS) and disease-free survival (DFS) in papillary RCC. In addition, a positive correlation between P4HA1 expression and immune cell infiltration was observed in clear cell RCC. We also identified a strong correlation between P4HA1 expression and immune checkpoint gene expression, microsatellite instability, and tumor mutation burden in chromophobe RCC. Finally, the results of in vitro experiments verified that overexpression of P4HA1 promoted the proliferation, migration, invasion, and epithelial-mesenchymal transition of RCC cells. Conclusion: Overall, our study has suggested that P4HA1 might play a significant role in tumorigenesis in RCC and may be a prognostic biomarker and therapeutic target for several malignant tumors, including RCC.