The Flt3-inhibitor quizartinib augments apoptosis and promotes maladaptive remodeling after myocardial infarction in mice.

BACKGROUND: Tyrosine kinase inhibitors (TKIs) targeting fms-like tyrosine kinase 3 (Flt3) such as quizartinib were specifically designed for acute myeloid leukemia treatment, but also multi-targeting TKIs applied to solid tumor patients inhibit Flt3. Flt3 is expressed in the heart and its activation is cytoprotective in myocardial infarction (MI) in mice. OBJECTIVES: We sought to test whether Flt3-targeting TKI treatment aggravates cardiac injury after MI. METHODS AND RESULTS: Compared to vehicle, quizartinib (10 mg/kg/day, gavage) did not alter cardiac dimensions or function in healthy mice after four weeks of therapy. Pretreated mice were randomly assigned to MI or sham surgery while receiving quizartinib or vehicle for one more week. Quizartinib did not aggravate the decline in ejection fraction, but significantly enhanced ventricular dilatation one week after infarction. In addition, apoptotic cell death was significantly increased in the myocardium of quizartinib-treated compared to vehicle-treated mice. In vitro, quizartinib dose-dependently decreased cell viability in neonatal rat ventricular myocytes and in H9c2 cells, and increased apoptosis as assessed in the latter. Together with H2O2, quizartinib potentiated the phosphorylation of the pro-apoptotic mitogen activated protein kinase p38 and augmented H2O2-induced cell death and apoptosis beyond additive degree. Pretreatment with a p38 inhibitor abolished apoptosis under quizartinib and H2O2. CONCLUSION: Quizartinib potentiates apoptosis and promotes maladaptive remodeling after MI in mice at least in part via a p38-dependent mechanism. These findings are consistent with the multi-hit hypothesis of cardiotoxicity and make cardiac monitoring in patients with ischemic heart disease under Flt3- or multi-targeting TKIs advisable.

C/EBPß isoform-specific regulation of podocyte pyroptosis in lupus nephritis-induced renal injury.

As an essential factor in the prognosis of systemic lupus erythematosus (SLE), lupus nephritis (LN) can accelerate the rate at which patients with SLE can transition to chronic kidney disease or even end-stage renal disease. Podocytes now appear to be a possible direct target in LN in addition to being prone to collateral damage from glomerular capillary lesions induces by immune complexes and inflammatory processes. The NLRP3 inflammasome is regulated by CCAAT/enhancer-binding protein ß (C/EBPß), which is involved in the pathogenesis of SLE. However, the role and mechanism of C/EBPß in LN remain unclear. In this investigation, glomerular podocytes treated with LN serum and MRL/lpr mice were employed as in vivo and in vitro models of LN, respectively. In vivo, the expression of C/EBPß isoforms was detected in kidney specimens of humans and mice with LN. Then we assessed the effect of C/EBPß inhibition on renal structure and function by injecting RNAi adeno-associated virus of C/EBPß shRNA into MRL/lpr mice. In vitro, glomerular podocytes were treated with LN serum and C/EBPß siRNA to explore the role of C/EBPß in the activation of the AIM2 inflammasome and podocyte injury. C/EBPß-LAP and C/EBPß-LIP were significantly overexpressed in kidney tissue samples from LN patients and mice, and C/EBPß inhibition significantly alleviated renal function damage and ameliorated renal structural deficiencies. Inflammatory pathways downstream from the AIM2 inflammasome could be suppressed by C/EBPß knockdown. Furthermore, the upregulation of C/EBPß-LAP could activate the AIM2 inflammasome and podocyte pyroptosis by binding to the promoters of AIM2 and CASPASE1 to enhance their expression, and the knockdown of AIM2 or (and) caspase-1 reversed the effects of C/EBPß-LAP overexpression. Interestingly, C/EBPß-LIP overexpression could transcriptionally inhibit IRAG and promote Ca2+ release-mediated activation of the AIM2 inflammasome. This finding suggests that C/EBPß is not only involved in the regulation of the expression of key proteins of the AIM2 inflammasome but also affects the polymerization of key proteins of the AIM2 inflammasome through the regulation of Ca2+ release. In conclusion, this study provides a new idea for studying the regulatory mechanism of C/EBPß and provides a theoretical basis for the early diagnosis and treatment of LN in the future. © 2023 The Pathological Society of Great Britain and Ireland.

Defective Desmosomal Adhesion Causes Arrhythmogenic Cardiomyopathy by Involving an Integrin-αVß6/TGF-ß Signaling Cascade.

BACKGROUND: Arrhythmogenic cardiomyopathy (ACM) is characterized by progressive loss of cardiomyocytes with fibrofatty tissue replacement, systolic dysfunction, and life-threatening arrhythmias. A substantial proportion of ACM is caused by mutations in genes of the desmosomal cell-cell adhesion complex, but the underlying mechanisms are not well understood. In the current study, we investigated the relevance of defective desmosomal adhesion for ACM development and progression. METHODS: We mutated the binding site of DSG2 (desmoglein-2), a crucial desmosomal adhesion molecule in cardiomyocytes. This DSG2-W2A mutation abrogates the tryptophan swap, a central interaction mechanism of DSG2 on the basis of structural data. Impaired adhesive function of DSG2-W2A was confirmed by cell-cell dissociation assays and force spectroscopy measurements by atomic force microscopy. The DSG2-W2A knock-in mouse model was analyzed by echocardiography, ECG, and histologic and biomolecular techniques including RNA sequencing and transmission electron and superresolution microscopy. The results were compared with ACM patient samples, and their relevance was confirmed in vivo and in cardiac slice cultures by inhibitor studies applying the small molecule EMD527040 or an inhibitory integrin-αVß6 antibody. RESULTS: The DSG2-W2A mutation impaired binding on molecular level and compromised intercellular adhesive function. Mice bearing this mutation develop a severe cardiac phenotype recalling the characteristics of ACM, including cardiac fibrosis, impaired systolic function, and arrhythmia. A comparison of the transcriptome of mutant mice with ACM patient data suggested deregulated integrin-αVß6 and subsequent transforming growth factor-ß signaling as driver of cardiac fibrosis. Blocking integrin-αVß6 led to reduced expression of profibrotic markers and reduced fibrosis formation in mutant animals in vivo. CONCLUSIONS: We show that disruption of desmosomal adhesion is sufficient to induce a phenotype that fulfils the clinical criteria to establish the diagnosis of ACM, confirming the dysfunctional adhesion hypothesis. Deregulation of integrin-αVß6 and transforming growth factor-ß signaling was identified as a central step toward fibrosis. A pilot in vivo drug test revealed this pathway as a promising target to ameliorate fibrosis. This highlights the value of this model to discern mechanisms of cardiac fibrosis and to identify and test novel treatment options for ACM.

Ultrasound Molecular Imaging of Atherosclerosis With Nanobodies: Translatable Microbubble Targeting Murine and Human VCAM (Vascular Cell Adhesion Molecule) 1.

OBJECTIVE: Contrast-enhanced ultrasound molecular imaging (CEUMI) of endothelial expression of VCAM (vascular cell adhesion molecule)-1 could improve risk stratification for atherosclerosis. The microbubble contrast agents developed for preclinical studies are not suitable for clinical translation. Our aim was to characterize and validate a microbubble contrast agent using a clinically translatable single-variable domain immunoglobulin (nanobody) ligand. Approach and Results: Microbubble with a nanobody targeting VCAM-1 (MBcAbVcam1-5) and microbubble with a control nanobody (MBVHH2E7) were prepared and characterized in vitro. Attachment efficiency to VCAM-1 under continuous and pulsatile flow was investigated using activated murine endothelial cells. In vivo CEUMI of the aorta was performed in atherosclerotic double knockout and wild-type mice after injection of MBcAbVcam1-5 and MBVHH2E7. Ex vivo CEUMI of human endarterectomy specimens was performed in a closed-loop circulation model. The surface density of the nanobody ligand was 3.5×105 per microbubble. Compared with MBVHH2E7, MBcAbVcam1-5 showed increased attachment under continuous flow with increasing shear stress of 1-8 dynes/cm2 while under pulsatile flow attachment occurred at higher shear stress. CEUMI in double knockout mice showed signal enhancement for MBcAbVcam1-5 in early (P=0.0003 versus MBVHH2E7) and late atherosclerosis (P=0.007 versus MBVHH2E7); in wild-type mice, there were no differences between MBcAbVcam1-5 and MBVHH2E7. CEUMI in human endarterectomy specimens showed a 100% increase in signal for MBcAbVcam1-5versus MBVHH2E7 (20.6±27.7 versus 9.6±14.7, P=0.0156). CONCLUSIONS: CEUMI of the expression of VCAM-1 is feasible in murine models of atherosclerosis and on human tissue using a clinically translatable microbubble bearing a VCAM-1 targeted nanobody.

mTOR, cardiomyocytes and inflammation in cardiac hypertrophy.

Mammalian target of rapamycin (mTOR) is an evolutionary conserved kinase that senses the nutrient and energy status of cells, the availability of growth factors, stress stimuli and other cellular and environmental cues. It responds by regulating a range of cellular processes related to metabolism and growth in accordance with the available resources and intracellular needs. mTOR has distinct functions depending on its assembly in the structurally distinct multiprotein complexes mTORC1 or mTORC2. Active mTORC1 enhances processes including glycolysis, protein, lipid and nucleotide biosynthesis, and it inhibits autophagy. Reported functions for mTORC2 after growth factor stimulation are very diverse, are tissue and cell-type specific, and include insulin-stimulated glucose transport and enhanced glycogen synthesis. In accordance with its cellular functions, mTOR has been demonstrated to regulate cardiac growth in response to pressure overload and is also known to regulate cells of the immune system. The present manuscript presents recently obtained insights into mechanisms whereby mTOR may change anabolic, catabolic and stress response pathways in cardiomocytes and discusses how mTOR may affect inflammatory cells in the heart during hemodynamic stress. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.

Neuregulin-1ß promotes glucose uptake via PI3K/Akt in neonatal rat cardiomyocytes.

Nrg1ß is critically involved in cardiac development and also maintains function of the adult heart. Studies conducted in animal models showed that it improves cardiac performance under a range of pathological conditions, which led to its introduction in clinical trials to treat heart failure. Recent work also implicated Nrg1ß in the regenerative potential of neonatal and adult hearts. The molecular mechanisms whereby Nrg1ß acts in cardiac cells are still poorly understood. In the present study, we analyzed the effects of Nrg1ß on glucose uptake in neonatal rat ventricular myocytes and investigated to what extent mTOR/Akt signaling pathways are implicated. We show that Nrg1ß enhances glucose uptake in cardiomyocytes as efficiently as IGF-I and insulin. Nrg1ß causes phosphorylation of ErbB2 and ErbB4 and rapidly induces the phosphorylation of FAK (Tyr(861)), Akt (Thr(308) and Ser(473)), and its effector AS160 (Thr(642)). Knockdown of ErbB2 or ErbB4 reduces Akt phosphorylation and blocks the glucose uptake. The Akt inhibitor VIII and the PI3K inhibitors LY-294002 and Byl-719 abolish Nrg1ß-induced phosphorylation and glucose uptake. Finally, specific mTORC2 inactivation after knockdown of rictor blocks the Nrg1ß-induced increases in Akt-p-Ser(473) but does not modify AS160-p-Thr(642) or the glucose uptake responses to Nrg1ß. In conclusion, our study demonstrates that Nrg1ß enhances glucose uptake in cardiomyocytes via ErbB2/ErbB4 heterodimers, PI3Kα, and Akt. Furthermore, although Nrg1ß activates mTORC2, the resulting Akt-Ser(473) phosphorylation is not essential for glucose uptake induction. These new insights into pathways whereby Nrg1ß regulates glucose uptake in cardiomyocytes may contribute to the understanding of its regenerative capacity and protective function in heart failure.

2-Substituted 3ß-Aryltropane Cocaine Analogs Produce Atypical Effects without Inducing Inward-Facing Dopamine Transporter Conformations.

Previous structure-activity relationship studies indicate that a series of cocaine analogs, 3ß-aryltropanes with 2ß-diarylmethoxy substituents, selectively bind to the dopamine transporter (DAT) with nanomolar affinities that are 10-fold greater than the affinities of their corresponding 2α-enantiomers. The present study compared these compounds to cocaine with respect to locomotor effects in mice, and assessed their ability to substitute for cocaine (10 mg/kg, i.p.) in rats trained to discriminate cocaine from saline. Despite nanomolar DAT affinity, only the 2ß-Ph2COCH2-3ß-4-Cl-Ph analog fully substituted for cocaine-like discriminative effects. Whereas all of the 2ß compounds increased locomotion, only the 2ß-(4-ClPh)PhCOCH2-3ß-4-Cl-Ph analog had cocaine-like efficacy. None of the 2α-substituted compounds produced either of these cocaine-like effects. To explore the molecular mechanisms of these drugs, their effects on DAT conformation were probed using a cysteine-accessibility assay. Previous reports indicate that cocaine binds with substantially higher affinity to the DAT in its outward (extracellular)- compared with inward-facing conformation, whereas atypical DAT inhibitors, such as benztropine, have greater similarity in affinity to these conformations, and this is postulated to explain their divergent behavioral effects. All of the 2ß- and 2α-substituted compounds tested altered cysteine accessibility of DAT in a manner similar to cocaine. Furthermore, molecular dynamics of in silico inhibitor-DAT complexes suggested that the 2-substituted compounds reach equilibrium in the binding pocket in a cocaine-like fashion. These behavioral, biochemical, and computational results show that aryltropane analogs can bind to the DAT and stabilize outward-facing DAT conformations like cocaine, yet produce effects that differ from those of cocaine.

Molecular imaging reveals rapid reduction of endothelial activation in early atherosclerosis with apocynin independent of antioxidative properties.

OBJECTIVE: Antioxidative drugs continue to be developed for the treatment of atherosclerosis. Apocynin is an nicotinamide adenine dinucleotide phosphate oxidase inhibitor with anti-inflammatory properties. We used contrast-enhanced ultrasound molecular imaging to assess whether short-term apocynin therapy in atherosclerosis reduces vascular oxidative stress and endothelial activation APPROACH AND RESULTS: Genetically modified mice with early atherosclerosis were studied at baseline and after 7 days of therapy with apocynin (4 mg/kg per day IP) or saline. Contrast-enhanced ultrasound molecular imaging of the aorta was performed with microbubbles targeted to vascular cell adhesion molecule 1 (VCAM-1; MB(V)), to platelet glycoprotein Ibα (MB(Pl)), and control microbubbles (MB(Ctr)). Aortic vascular cell adhesion molecule 1 was measured using Western blot. Aortic reactive oxygen species generation was measured using a lucigenin assay. Hydroethidine oxidation was used to assess aortic superoxide generation. Baseline signal for MBV (1.3 ± 0.3 AU) and MB(Pl )(1.5 ± 0.5 AU) was higher than for MBCtr (0.5 ± 0.2 AU; P<0.01). In saline-treated animals, signal did not significantly change for any microbubble agent, whereas short-term apocynin significantly (P<0.05) reduced vascular cell adhesion molecule 1 and platelet signal (MBV: 0.3 ± 0.1; MBPl: 0.4 ± 0.1; MBCtr: 0.3 ± 0.2 AU; P=0.6 between agents). Apocynin reduced aortic vascular cell adhesion molecule 1 expression by 50% (P<0.05). However, apocynin therapy did not reduce reactive oxygen species content, superoxide generation, or macrophage content. CONCLUSIONS: Short-term treatment with apocynin in atherosclerosis reduces endothelial cell adhesion molecule expression. This change in endothelial phenotype can be detected by molecular imaging before any measurable decrease in macrophage content and is not associated with a detectable change in oxidative burden.

Identification of potential key lipid metabolism-related genes involved in tubular injury in diabetic kidney disease by bioinformatics analysis.

AIMS: Accumulating evidences indicate that abnormalities in tubular lipid metabolism play a crucial role in the development of diabetic kidney disease (DKD). We aim to identify novel lipid metabolism-related genes associated with tubular injury in DKD by utilizing bioinformatics approaches. METHODS: Differentially expressed genes (DEGs) between control and DKD tubular tissue samples were screened from the Gene Expression Omnibus (GEO) database, and then were intersected with lipid metabolism-related genes. Hub genes were further determined by combined weighted gene correlation network analysis (WGCNA) and protein-protein interaction (PPI) network. We performed enrichment analysis, immune analysis, clustering analysis, and constructed networks between hub genes and miRNAs, transcription factors and small molecule drugs. Receiver operating characteristic (ROC) curves were employed to evaluate the diagnostic efficacy of hub genes. We validated the relationships between hub genes and DKD with external datasets and our own clinical samples. RESULTS: There were 5 of 37 lipid metabolism-related DEGs identified as hub genes. Enrichment analysis demonstrated that lipid metabolism-related DEGs were enriched in pathways such as peroxisome proliferator-activated receptors (PPAR) signaling and pyruvate metabolism. Hub genes had potential regulatory relationships with a variety of miRNAs, transcription factors and small molecule drugs, and had high diagnostic efficacy. Immune infiltration analysis revealed that 13 immune cells were altered in DKD, and hub genes exhibited significant correlations with a variety of immune cells. Through clustering analysis, DKD patients could be classified into 3 immune subtypes and 2 lipid metabolism subtypes, respectively. The tubular expression of hub genes in DKD was further verified by other external datasets, and immunohistochemistry (IHC) staining showed that except ACACB, the other 4 hub genes (LPL, AHR, ME1 and ALOX5) exhibited the same results as the bioinformatics analysis. CONCLUSION: Our study identified several key lipid metabolism-related genes (LPL, AHR, ME1 and ALOX5) that might be involved in tubular injury in DKD, which provide new insights and perspectives for exploring the pathogenesis and potential therapeutic targets of DKD.

Postoperative encapsulated hemoperitoneum in a patient with gastric stromal tumor treated by exposed endoscopic full-thickness resection: A case report.

BACKGROUND: Gastric stromal tumors, originating from mesenchymal tissues, are one of the most common tumors of the digestive tract. For stromal tumors originating from the muscularis propria, compared with conventional endoscopic submucosal dissection (ESD), endoscopic full-thickness resection (EFTR) can remove deep lesions and digestive tract wall tumors completely. However, this technique has major limitations such as perforation, postoperative bleeding, and post-polypectomy syndrome. Herein, we report a case of postoperative serous surface bleeding which formed an encapsulated hemoperitoneum in a patient with gastric stromal tumor that was treated with exposed EFTR. Feasible treatment options to address this complication are described. CASE SUMMARY: A 47-year-old male patient had a hemispherical protrusion found during gastric endoscopic ultrasonography, located at the upper gastric curvature adjacent to the stomach fundus, with a smooth surface mucosa and poor mobility. The lesion was 19.3 mm × 16.1 mm in size and originated from the fourth ultrasound layer. Computed tomography (CT) revealed no significant evidence of lymph node enlargement or distant metastasis. Using conventional ESD technology for mucosal pre-resection, exposed EFTR was performed to resect the intact tumor in order to achieve a definitive histopathological diagnosis. Based on its morphology and immunohistochemical expression of CD117 and DOG-1, the lesion was proven to be consistent with a gastric stromal tumor. Six days after exposed EFTR, CT showed a large amount of encapsulated fluid and gas accumulation around the stomach. In addition, gastroscopy suggested intracavitary bleeding and abdominal puncture drainage indicated serosal bleeding. Based on these findings, the patient was diagnosed with serosal bleeding resulting in encapsulated abdominal hemorrhage after exposed EFTR for a gastric stromal tumor. The patient received combined treatments, such as hemostasis under gastroscopy, gastrointestinal decompression, and abdominal drainage. All examinations were normal within six months of follow-up. CONCLUSION: This patient developed serous surface bleeding in the gastric cavity following exposed EFTR. Serosal bleeding resulting in an encapsulated hemoperitoneum is rare in clinical practice. The combined treatment may replace certain surgical techniques.

Liraglutide Lowers Endothelial Vascular Cell Adhesion Molecule-1 in Murine Atherosclerosis Independent of Glucose Levels.

The authors determined the effect of the GLP-1 receptor agonist liraglutide on endothelial surface expression of vascular cell adhesion molecule (VCAM)-1 in murine apolipoprotein E knockout atherosclerosis. Contrast-enhanced ultrasound molecular imaging using microbubbles targeted to VCAM-1 and control microbubbles showed a 3-fold increase in endothelial surface VCAM-1 signal in vehicle-treated animals, whereas in the liraglutide-treated animals the signal ratio remained around 1 throughout the study. Liraglutide had no influence on low-density lipoprotein cholesterol or glycated hemoglobin, but reduced TNF-α, IL-1ß, MCP-1, and OPN. Aortic plaque lesion area and luminal VCAM-1 expression on immunohistology were reduced under liraglutide treatment.

Accelerating Wound Closure With Metrnl in Normal and Diabetic Mouse Skin.

Impaired wound healing and ulcer complications are major causes of morbidity in patients with diabetes. Impaired wound healing is associated with increased inflammation and poor angiogenesis in diabetes patients. Here, we demonstrate that topical administration of a secreted recombinant protein (Meteorin-like [Metrnl]) accelerates wound epithelialization and angiogenesis in mice. We observed a significant increase in Metrnl expression during physiological wound healing; however, its expression remained low during diabetic wound healing. Functionally, the recombinant protein Metrnl significantly accelerated wound closure in normal and diabetic mice models including db/db, high-fat diet/streptozotocin (HFD/STZ), and STZ mice. Mechanistically, keratinocytes secrete quantities of Metrnl to promote angiogenesis; increase endothelial cell proliferation, migration, and tube formation; and enhance macrophage polarization to the M2 type. Meanwhile, M2 macrophages secrete Metrnl to further stimulate angiogenesis. Moreover, the keratinocyte- and macrophage-produced cytokine Metrnl drives postinjury angiogenesis and reepithelialization through activation of AKT phosphorylation (S473) in a KIT receptor tyrosine kinase (c-Kit)-dependent manner. In conclusion, our study suggests that Metrnl has a biological effect in accelerating wound closure through c-Kit-dependent angiogenesis and epithelialization.

The UDPase ENTPD5 regulates ER stress-associated renal injury by mediating protein N-glycosylation.

Impaired protein N-glycosylation leads to the endoplasmic reticulum (ER) stress, which triggers adaptive survival or maladaptive apoptosis in renal tubules in diabetic kidney disease (DKD). Therapeutic strategies targeting ER stress are promising for the treatment of DKD. Here, we report a previously unappreciated role played by ENTPD5 in alleviating renal injury by mediating ER stress. We found that ENTPD5 was highly expressed in normal renal tubules; however, ENTPD5 was dynamically expressed in the kidney and closely related to pathological DKD progression in both human patients and mouse models. Overexpression of ENTPD5 relieved ER stress in renal tubular cells, leading to compensatory cell proliferation that resulted in hypertrophy, while ENTPD5 knockdown aggravated ER stress to induce cell apoptosis, leading to renal tubular atrophy and interstitial fibrosis. Mechanistically, ENTPD5-regulated N-glycosylation of proteins in the ER to promote cell proliferation in the early stage of DKD, and continuous hyperglycemia activated the hexosamine biosynthesis pathway (HBP) to increase the level of UDP-GlcNAc, which driving a feedback mechanism that inhibited transcription factor SP1 activity to downregulate ENTPD5 expression in the late stage of DKD. This study was the first to demonstrate that ENTPD5 regulated renal tubule cell numbers through adaptive proliferation or apoptosis in the kidney by modulating the protein N-glycosylation rate in the ER, suggesting that ENTPD5 drives cell fate in response to metabolic stress and is a potential therapeutic target for renal diseases.

Metrnl Alleviates Lipid Accumulation by Modulating Mitochondrial Homeostasis in Diabetic Nephropathy.

Ectopic lipid accumulation in renal tubules is closely related to the pathogenesis of diabetic kidney disease (DKD), and mitochondrial dysfunction is thought to play a key role in lipid accumulation. Therefore, maintaining mitochondrial homeostasis holds considerable promise as a therapeutic strategy for the treatment of DKD. Here, we report that the Meteorin-like (Metrnl) gene product mediates lipid accumulation in the kidney and has therapeutic potential for DKD. We confirmed the reduced expression of Metrnl in renal tubules, which was inversely correlated with DKD pathological changes in human patients and mouse models. Functionally, pharmacological administration of recombinant Metrnl (rMetrnl) or Metrnl overexpression could alleviate lipid accumulation and inhibit kidney failure. In vitro, rMetrnl or Metrnl overexpression attenuated palmitic acid-induced mitochondrial dysfunction and lipid accumulation in renal tubules accompanied by maintained mitochondrial homeostasis and enhanced lipid consumption. Conversely, shRNA-mediated Metrnl knockdown diminished the protective effect on the kidney. Mechanistically, these beneficial effects of Metrnl were mediated by the Sirt3-AMPK signaling axis to maintain mitochondrial homeostasis and through Sirt3-uncoupling protein-1 to promote thermogenesis, consequently alleviating lipid accumulation. In conclusion, our study demonstrates that Metrnl regulated lipid metabolism in the kidney by modulating mitochondrial function and is a stress-responsive regulator of kidney pathophysiology, which sheds light on novel strategies for treating DKD and associated kidney diseases. ARTICLE HIGHLIGHTS: Metrnl is expressed in renal tubules and is reduced under diabetic conditions. The concentration of Metrnl in the kidney is correlated with lipid accumulation and serum creatinine. Metrnl-specific overexpression in the kidney or recombinant Metrnl administration alleviates renal injuries in diabetic mice. Metrnl regulates renal tubules lipid metabolism through Sirt3-AMPK/UCP1 signaling axis-mediated mitochondrial homeostasis.

Application Effect of Laparoscopic Myomectomy and Comprehensive Rehabilitation Nursing on Patients with Uterine Fibroids.

Background: Uterine fibroids are most common in women aged 30-50 and are the most common benign gynecological tumors. Relevant data suggest that about 25% of patients with uterine fibroids are at childbearing age. Uterine fibroids not only cause the discomfort symptoms, and affect the pregnancy, but also have certain malignant transformation risk, thus needed to be treated positively and promptly. Aim: This study is aimed at exploring the effect of laparoscopic myomectomy and comprehensive rehabilitation nursing on patients with uterine fibroids. Methods: The clinical data of 110 cases of uterine fibroids admitted to our hospital from August 2019 to December 2021 were analyzed retrospectively, and they were divided into two groups according to postoperative rehabilitation strategies. Both groups were treated with laparoscopic myomectomy. The A group was treated with routine rehabilitation strategy, while the B group was treated with comprehensive rehabilitation nursing strategy. The differences in operation-related indicators, stress factors, inflammatory factors, nutritional indicators, knowledge mastery, occurrence of adverse symptoms and pain scores, negative emotion scores, nursing satisfaction, and simplified comfort status scale (GCQ) scores between the two groups under nursing strategies were compared. Results: The postoperative exhaust time (13.14 ± 2.03) h, bed time (9.86 ± 1.94) h, postoperative hospital stay (4.37 ± 1.31) d, and total hospital stay (6.78 ± 1.69) d in the B group were shorter than those in the A group, and the hospitalization expenses (0.74 ± 0.25) million were less than those in the A group (P < 0.05). Before operation, stress factors, inflammatory factors, and nutritional indexes were compared between the two groups (P > 0.05). On the 3rd day after operation, tumor necrosis factor-α (TNF-α), cortisol (Cor), norepinephrine (NE), and interleukin-1ß (IL-1ß) in the two groups showed a significantly upward trend compared with those before operation, and albumin and transferrin were significantly fell compared with those before operation. However, the values of stress factor and inflammatory factor in the B group were significantly lower than those in the A group, and the values after the decrease of nutritional index were significantly higher than those in the A group (P < 0.05). The pain scores at 24 h, 48 h, and 72 h after operation in the B group were significantly lower than those in the A group (P < 0.05). Negative emotions, nursing satisfaction, and GCQ scores were compared between the two groups before intervention (P > 0.05). After the intervention, the scores of Hamilton Depression Scale (HAMD) and Hamilton Anxiety Scale (HAMA) in the two groups were significantly lower than those before the intervention, and the scores of nursing satisfaction and GCQ were higher than those before the intervention. The values of negative emotions in the B group after the decline were significantly lower than those in the A group, while the values of nursing satisfaction and GCQ after the increase were higher than those in the A group (P < 0.05). The excellent and good rate of knowledge acquisition in the B group was 94.55% (52/55), which was significantly higher than 78.18% (43/55) in the A group (P < 0.05). The incidence of adverse symptoms in the B group was 9.09% (5/55), which was lower than 21.82% (12/55) in the A group, while the difference was not statistically significant (P > 0.05). Conclusion: Laparoscopic myomectomy combined with comprehensive rehabilitation nursing can reduce the postoperative stress state of patients with uterine fibroids, improve patient satisfaction, reduce adverse emotions, and promote rehabilitation.

Fms-like tyrosine kinase 3 is a regulator of the cardiac side population in mice.

Fms-like tyrosine kinase 3 (Flt3) is a regulator of hematopoietic progenitor cells and a target of tyrosine kinase inhibitors. Flt3-targeting tyrosine kinase inhibitors can have cardiovascular side effects. Flt3 and its ligand (Flt3L) are expressed in the heart, but little is known about their physiological functions. Here, we show that cardiac side population progenitor cells (SP-CPCs) from mice produce and are responsive to Flt3L. Compared with wild-type, flt3L-/- mice have less SP-CPCs with less contribution of CD45-CD34+ cells and lower expression of genes related to epithelial-to-mesenchymal transition, cardiovascular development and stem cell differentiation. Upon culturing, flt3L-/- SP-CPCs show increased proliferation and less vasculogenic commitment, whereas Akt phosphorylation is lower. Notably, proliferation and differentiation can be partially restored towards wild-type levels in the presence of alternative receptor tyrosine kinase-activating growth factors signaling through Akt. The lower vasculogenic potential of flt3L-/- SP-CPCs reflects in decreased microvascularisation and lower systolic function of flt3L-/- hearts. Thus, Flt3 regulates phenotype and function of murine SP-CPCs and contributes to cellular and molecular properties that are relevant for their cardiovasculogenic potential.

Loss of MEN1 leads to renal fibrosis and decreases HGF-Adamts5 pathway activity via an epigenetic mechanism.

BACKGROUND: Renal fibrosis is a serious condition that results in the development of chronic kidney diseases. The MEN1 gene is an epigenetic regulator that encodes the menin protein and its role in kidney tissue remains unclear. METHODS: Kidney histology was examined on paraffin sections stained with hematoxylin-eosin staining. Masson's trichrome staining and Sirius red staining were used to analyze renal fibrosis. Gene and protein expression were determined by quantitative real-time PCR (qPCR) and Western blot, respectively. Immunohistochemistry staining in the kidney tissues from mice or patients was used to evaluate protein levels. Flow cytometry was used to analyze the cell cycle distributions and apoptosis. RNA-sequencing was performed for differential expression genes in the kidney tissues of the Men1f/f and Men1∆/∆ mice. Chromatin immunoprecipitation sequencing (ChIP-seq) was carried out for identification of menin- and H3K4me3-enriched regions within the whole genome in the mouse kidney tissue. ChIP-qPCR assays were performed for occupancy of menin and H3K4me3 at the gene promoter regions. Luciferase reporter assay was used to detect the promoter activity. The exacerbated unilateral ureteral obstruction (UUO) models in the Men1f/f and Men1∆/∆ mice were used to assess the pharmacological effects of rh-HGF on renal fibrosis. RESULTS: The expression of MEN1 is reduce in kidney tissues of fibrotic mouse and human diabetic patients and treatment with fibrotic factor results in the downregulation of MEN1 expression in renal tubular epithelial cells (RTECs). Disruption of MEN1 in RTECs leads to high expression of α-SMA and Collagen 1, whereas MEN1 overexpression restrains epithelial-to-mesenchymal transition (EMT) induced by TGF-ß treatment. Conditional knockout of MEN1 resulted in chronic renal fibrosis and UUO-induced tubulointerstitial fibrosis (TIF), which is associated with an increased induction of EMT, G2/M arrest and JNK signaling. Mechanistically, menin recruits and increases H3K4me3 at the promoter regions of hepatocyte growth factor (HGF) and a disintegrin and metalloproteinase with thrombospondin motifs 5 (Adamts5) genes and enhances their transcriptional activation. In the UUO mice model, exogenous HGF restored the expression of Adamts5 and ameliorated renal fibrosis induced by Men1 deficiency. CONCLUSIONS: These findings demonstrate that MEN1 is an essential antifibrotic factor in renal fibrogenesis and could be a potential target for antifibrotic therapy.

NOX1 mediates metabolic heart disease in mice and is upregulated in monocytes of humans with diastolic dysfunction.

AIMS: Microvascular inflammation plays an important role in the pathogenesis of diastolic dysfunction (DD) and metabolic heart disease. NOX1 is expressed in vascular and immune cells and has been implicated in the vascular pathology of metabolic disease. However, its contribution to metabolic heart disease is less understood. METHODS AND RESULTS: NOX1-deficient mice (KO) and male wild-type (WT) littermates were fed a high-fat high-sucrose diet (HFHS) and injected streptozotocin (75 mg/kg i.p.) or control diet (CTD) and sodium citrate. Despite similar weight gain and increase in fasting blood glucose and insulin, only WT-HFHS but not KO-HFHS mice developed concentric cardiac hypertrophy and elevated left ventricular filling pressure. This was associated with increased endothelial adhesion molecule expression, accumulation of Mac-2-, IL-1ß-, and NLRP3-positive cells and nitrosative stress in WT-HFHS but not KO-HFHS hearts. Nox1 mRNA was solidly expressed in CD45+ immune cells isolated from healthy mouse hearts but was negligible in cardiac CD31+ endothelial cells. However, in vitro, Nox1 expression increased in response to lipopolysaccharide (LPS) in endothelial cells and contributed to LPS-induced upregulation of Icam-1. Nox1 was also upregulated in mouse bone marrow-derived macrophages in response to LPS. In peripheral monocytes from age- and sex-matched symptomatic patients with and without DD, NOX1 was significantly higher in patients with DD compared to those without DD. CONCLUSIONS: NOX1 mediates endothelial activation and contributes to myocardial inflammation and remodelling in metabolic disease in mice. Given its high expression in monocytes of humans with DD, NOX1 may represent a potential target to mitigate heart disease associated with DD.

Designed Ankyrin Repeat Proteins as Novel Binders for Ultrasound Molecular Imaging.

Clinical translation of ultrasound molecular imaging will depend on the development of binders that can easily be generated, manufactured and coupled, and that are compatible with in vivo use. We describe targeted microbubbles (MBs) using designed ankyrin repeat proteins (DARPins) as a novel class of such translatable binders. Candidate DARPin binders for vascular cell adhesion molecule 1, an endothelial cell adhesion molecule involved in inflammatory processes, were selected using ribosome display and coupled to MBs. Flow-chamber assays of five MBs carrying high-affinity binders showed selective retention on endothelial cells activated by tumor necrosis factor-α for two binders compared with a MB carrying a control DARPin. In vivo ultrasound molecular imaging in a murine hind-limb inflammation model demonstrated up to a fourfold signal enhancement for three of the five MBs versus control. However, there was no correlation between results from flow-chamber assays and in vivo imaging. Thus, we conclude that ultrasound molecular imaging of inflammation using DARPin binders is feasible per se, but that screening of candidates cannot be accomplished with flow-chamber assays as used in our study.

Neuregulin-1 triggers GLUT4 translocation and enhances glucose uptake independently of insulin receptor substrate and ErbB3 in neonatal rat cardiomyocytes.

During stress conditions such as pressure overload and acute ischemia, the myocardial endothelium releases neuregulin-1ß (NRG-1), which acts as a cardioprotective factor and supports recovery of the heart. Recently, we demonstrated that recombinant human (rh)NRG-1 enhances glucose uptake in neonatal rat ventricular myocytes via the ErbB2/ErbB4 heterodimer and PI3Kα. The present study aimed to further elucidate the mechanism whereby rhNRG-1 activates glucose uptake in comparison to the well-established insulin and to extend the findings to adult models. Combinations of rhNRG-1 with increasing doses of insulin did not yield any additive effect on glucose uptake measured as 3H-deoxy-d-glucose incorporation, indicating that the mechanisms of the two stimuli are similar. In c-Myc-GLUT4-mCherry-transfected neonatal rat cardiomyocytes, rhNRG-1 increased sarcolemmal GLUT4 by 16-fold, similar to insulin. In contrast to insulin, rhNRG-1 did not phosphorylate IRS-1 at Tyr612, indicating that IRS-1 is not implicated in the signal transmission. Treatment of neonatal rats with rhNRG-1 induced a signaling response comparable with that observed in vitro, including increased ErbB4-pTyr1284, Akt-pThr308 and Erk1/2-pThr202/Tyr204. In contrast, in adult cardiomyocytes rhNRG-1 only increased the phosphorylation of Erk1/2 without having any significant effect on Akt and AS160 phosphorylation and glucose uptake, suggesting that rhNRG-1 function in neonatal cardiomyocytes differs from that in adult cardiomyocytes. In conclusion, our results show that similar to insulin, rhNRG-1 can induce glucose uptake by activating the PI3Kα-Akt-AS160 pathway and GLUT4 translocation. Unlike insulin, the rhNRG-1-induced effect is not mediated by IRS proteins and is observed in neonatal, but not in adult rat cardiomyocytes.