Histological Analysis Research Articles

Cholecystokinin-expressing neurons of the ventromedial hypothalamic nucleus control energy homeostasis

The hypothalamus is the primary center of the brain that regulates energy homeostasis. The ventromedial hypothalamus (VMH) plays a central role in maintaining energy balance by regulating food intake, energy expenditure, and glucose levels. However, the cellular and molecular mechanisms underlying its functions are still poorly understood. Cholecystokinin (CCK) is one of many genes expressed in this hypothalamic nucleus. Peripheral CCK regulates food intake, body weight, and glucose homeostasis. However, current research does not explain the function of CCK neurons in specific nuclei of the hypothalamus and their likely roles in network dynamics related to energy balance and food intake. This study uses genetic and pharmacological methods to examine the role of CCK-expressing neurons in the VMH (CCKVMH). Namely, using a previously generated BAC transgenic line expressing Cre recombinase under the CCK promoter, we performed targeted manipulations of CCKVMH neurons. Histological and transcriptomic database analysis revealed extensive distribution of these neurons in the VMH, with significant heterogeneity in gene expression related to energy balance, including co-expression with PACAP and somatostatin. Pharmacogenetic acute inhibition via Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) resulted in increased food intake and altered meal patterns, characterized by higher meal frequency and shorter intermeal intervals. Furthermore, diphtheria toxin-mediated ablation of CCKVMH neurons led to significant weight gain and hyperphagia over time, increasing meal size and duration. These mice also exhibited impaired glucose tolerance, indicative of disrupted glucose homeostasis. Our findings underscore the integral role of CCKVMH neurons in modulating feeding behavior, energy homeostasis, and glucose regulation. This study enhances our understanding of the neurohormonal mechanisms underlying obesity and metabolic disorders, providing potential targets for therapeutic interventions.

Novel Driver Genes and Mutations in Lung Pecoma: A Case Report

Introduction: Perivascular epithelioid cell tumors (PEComa) are rare mesenchymal neoplasms characterized by perivascular epithelioid cells. Despite their common occurrence in the uterus, gastrointestinal tract, and retroperitoneum, this study presents an exceptional case of PEComa identified in the lung, warranting unique molecular exploration. Case Report: A 50-year-old man was diagnosed with a 6 cm neoplasm in the lower lobe of the right lung without enlarged lymph nodes during a routine examination. Thoracotomy, extended lower bilobectomy, and D3 lymphadenectomy were performed. Histological and immunohistochemical analysis diagnosed a PEComa. No conventional TSC1 and TSC2 mutations specific to PEComa, which resulted in mTOR pathway activation, were detected by whole-exome sequencing. In contrast, mutations were unveiled in the MTOR, EIF4EBP1, and PRAME genes that could be an alternative mechanism governing mTOR activation. Conclusion: These findings provide novel insights into the molecular intricacies of lung PEComa, showcasing the distinctive roles of MTOR, EIF4EBP1, and PRAME mutations.

A personalized, 3D printed, polymeric elastic support device for the prevention of post-MI cardiac remodeling

Abstract Introduction Myocardial remodeling and dilatation following myocardial infarction (MI) initiates a vicious cycle leading to heart failure (HF). The anatomy and pathophysiology of every patient is unique. We have developed a 3D printed, biopolymer based personalized Elastic Support Device, that is 3D printed based on patient’s imaging, aiming to restore cardiac physiology. This study aimed to assess the device on rat MI model. Materials and Methods MI was induced in 24 Sprague Dawley rats by permanently ligating the mid LAD artery via midline sternotomy cut. Twelve animals underwent immediate implantation of the elastic support device that was pre-printed from animal specific 3D echocardiography. The animals were followed up with echocardiography at days -2, 7, 14, 28. The animals were sacrificed at day 28, cardiac tissue and plasma were taken for further histological and biochemical analysis. Results and discussion Mortality was similar at both groups (5 vs 4 in the device and control groups respectively). Ejection fraction reduced in both groups from a baseline of 61.9±8.9% to 47.5±12.7%. Left Ventricular End Diastolic Diameter increased from baseline to day 28 in the control (7.0±0.56mm vs 7.7±1mm) but not the device group (6.87±0.2mm vs 6.93±1.1mm, p<0.05). Parameters of diastolic function such as E/E’ were similar in both groups throughout the follow-up. Mason’s Trichrome stained cardiac sections revealed improved infarct size at the device group (7.4±1.9% vs. 10.6±2.8%) and lower overall cardiac fibrosis. Conclusion In this preliminary small animal model study, the capability to 3D print and implant an imaging based, personalized, 3D printed elastic support device. The cardiac diastolic function was not impaired, and no safety issues were noticed. Furthermore, cardiac dilatation was prevented, and infarct size was reduced.Trial design and results

Histological Analysis of Somatic Embryogenesis from Immature Zygotic Embryo of Wild Banana Musa acuminata ssp. malaccensis

Somatic embryogenesis, a crucial plant regeneration method, has become indispensable for crop improvement, particularly for species reliant on somatic cell manipulation techniques. Optimization of this process necessitates an understanding of the developmental stages involved. This study investigates the histological aspects of somatic embryogenesis in Musa acuminata ssp. malaccensis derived from immature zygotic embryos. Through detailed histological analysis, we aimed to elucidate the morphological changes and cellular organization occurring during the various stages of somatic embryogenesis, from induction, culture proliferation, and somatic embryo development to plantlet conversion. The initial stages of embryogenesis, characterized by nodules, were primarily composed of meristematic cells with high cell division activity. These cells contained tetrad-like structures that could develop into distinct two- and four-celled proembryoids or proembryogenic aggregates. Our histo-anatomical analysis revealed that embryogenic cultures proliferated through multiple pathways simultaneously: somatic embryo budding, proembryo formation, and pro-embryonic mass formation from both internal and peripheral cells. At the stage of somatic embryo development, embryos with a well-defined protoderm layer, containing cells with prominent nuclei and dense cytoplasm, potentially regenerate into plantlets. Furthermore, histological examination revealed the presence of procambium within mature somatic embryos, which subsequently developed into the vascular system of the complete plantlet

Long-acting CRF2 receptor agonist COR-1389 improves cardiopulmonary function parameters in monocrotaline-induced pulmonary hypertension and right heart failure rat model

Abstract Background Pulmonary hypertension (PH) associated with right heart failure (RHF) poses a significant therapeutic challenge. Short term studies have shown that urocortin-2 (UCN-2) administration, either intravenously or subcutaneously, can ameliorate cardiac and/or pulmonary parameters in human heart failure (HF) and in animal models of PH and RHF. Purpose This study aimed to evaluate the potential benefits of COR-1389, a long-acting CRF2 receptor agonist, on cardiopulmonary function and adverse remodeling in a rat model of monocrotaline (MCT)-induced PH and RHF. Method Monocrotaline (40 mg/kg) was administered subcutaneously in rats on Day-0 to induce PH and RHF, while the control group received vehicle. After 14 days, MCT-injected rats were evenly allocated into two groups based on echocardiography parameters of accelerated time/ejection time ratio and tricuspid annular plane systolic excursion (TAPSE), before receiving either COR-1389 (100 ug/kg) or its vehicle subcutaneously every 4 days for 14 days. On Day-28, echocardiography was repeated in all groups, followed by right heart catheterization to directly measure pulmonary hemodynamics. Heart and lung samples were prepared for histological analysis. Results By Day-28, MCT-treated rats exhibited significantly elevated mean pulmonary arterial pressure (mPAP) and right ventricular (RV) mass (Fulton Index/BW), along with decreased RV function (increased TAPSE) and reduced cardiac output (CO) compared to control rats. These changes were accompanied by severe tissue remodeling, including increased vascular muscularization (alpha-smooth muscle actin content) and pulmonary arterial wall thickening, RV fibrosis, and cardiomyocyte hypertrophy. Following 14 days of curative treatment, MCT+COR-1389 compared to MCT, significantly decreased mPAP and RV mass (Fulton Index/BW), while RV function improved (reduced TAPSE) and CO was increased. Moreover, COR-1389 attenuated adverse pulmonary vascular remodeling (reduced muscularization and arterial wall thickening), RV fibrosis, and cardiomyocyte hypertrophy, consistent with its hemodynamic effects. Conclusions Chronic CRF2 agonism with COR-1389 improved dysregulated cardiac and pulmonary function and structure in the MCT rat model, suggesting its potential for the treatment of PH and RHF.

Exosome-like nanovesicle targeting visceral adipose tissue inflammation mitigates heart failure with preserved ejection fraction

Abstract Introduction Heart failure (HF) with preserved ejection fraction (HFpEF) is associated with chronic systemic inflammation and obesity, with limited therapies. Inflammation in visceral adipose tissue (VAT) was significantly evoked in obese mice; while mesenchymal stem cell (MSC), especially atorvastatin (ATV)-pretreated MSC, could promote anti-inflammatory phenotype switch of immunocytes (e.g. macrophages), serving as a promising therapy for obesity-related HFpEF. Thus, the study was designed to prepare a novel ATV-pretreated MSC-derived exosome-like nanovesicles (ELNVs) and assess the anti-inflammatory and cardioprotective effects of ELNVs. Methods ELNVs derived from unpretreated MSC (ELNVM) or ATV-pretreated MSC (ELNVMA) were prepared and characterized (Figure 1). PKH-26-labelled ELNVs were incubated with THP1 cells for cell uptake assay. For anti-inflammatory studies in vitro, ELNVs (5×1010/mL) were added into the medium, followed by the induction of M1 or M2 polarization. The expressions of iNOS and Arg1 were measured by immunofluorescence (IF) staining. A 2-hit preclinical mice model of HFpEF, established by a 12-week high-fat diet (HFD) and Nω-Nitro-L-arginine methyl ester hydrochloride administration (0.5 g/L), was applied and received normal saline (NS) or ELNVs (i.p., 5×1011/kg body weight, 3 times a week). Functional and histological analysis including echocardiography, exhaustion test, WGA staining for hearts and western blot for VAT were performed. Quantitative polymerase chain reaction (qPCR) of key long non-coding RNAs (lncRNAs) was used to identify the mediator in ELNVs. Results The mean diameters of ELNVM and ELNVMA were 109.5 and 114.6 nm respectively and both ELNV types could be taken by THP1 cells. VAT inflammation was activated in HFpEF mice compared to the control in terms of the protein levels of iNOS and Arg1. An elevated expression of Arg1 and a decreased level of iNOS was observed in THP1 cells in vitro and VAT in vivo in both ELNV groups (P<0.05, Figure 1 and 2). Consistently, levels of interleukin (IL)-1β were increased in VAT of HFpEF mice, which were downregulated in ELNVM and ELNVMA groups. Both ELNVs could reduce the E/E’, and attenuate cardiac hypertrophy (heart weight to tibial length) and pulmonary congestion (lung weight [wet to dry]) (all P<0.05), with more pronounced improvements in ELNVMA group (Figure 2). Results of qPCR indicated three lncRNA candidates (TARID, H19 and KLF3-AS1) that were previously reported to inhibit M1 polarization or promote M2 polarization, were elevated in ELNVMA compared to ELNVM (P<0.05). Conclusion VAT inflammation was evoked in HFpEF mice and contributed to the development of HFpEF. This pilot study developed a novel ATV-pretreated ELNVs, which might serve as a promising tool for alleviating HFpEF due to the charming anti-inflammatory and cardioprotective effects mediated by regulating VAT macrophages.Figure 1.ELNVs’ PreparationFigure 2.Anti-inflammation of ELNVs

Unraveling Marfan hearts: ultrastructural evidence for myocardial remodeling and cardiomyopathy

Abstract Background Despite improved life expectancy for patients with Marfan syndrome (MFS), significant morbidity and mortality persist. Although primarily attributed to aortic dissection and rupture, recent studies highlight the emerging role of arrhythmias and heart failure as additional contributors to morbidity and mortality in MFS. While severe valvular heart disease is an established cause of heart failure, there is a growing recognition of primary cardiomyopathy in patients with MFS. The mechanisms underlying cardiomyopathy in MFS remain poorly understood. Purpose This study aimed to reveal insights into the ultrastructural myocardial architecture in patients with MFS, thereby elucidating the role of cardiomyopathy in MFS. Methods This study enrolled 44 patients who underwent cardiac surgery at 2 centers (Marfan syndrome patients n=27 (MFS), ascending thoracic aortic aneurysm repair patients n=5 (non-MFS), patients post-orthotopic heart transplant n=12 (controls)). Myocardial biopsy samples were subjected to histopathological analysis using transmission electron microscopy and conventional histology. Results Conventional histological analysis revealed subtle abnormalities in the cardiomyocyte morphology of MFS patients, compared to non-MFS and controls. Overall, a slight increase in interstitial fibrosis and mild myocyte hypertrophy were observed in MFS patients. Ultrastructural examination of both MFS and non-MFS patients revealed significant structural remodeling and degenerative changes in the myocardium. These changes included: extensive myofibril lysis, compromised cell membrane integrity and nuclear chromatin condensation. Degenerative lesions were often accompanied by cytoplasmic debris, lysosomal residual bodies, autophagic vacuoles, and severe mitochondrial pathology. Dense accumulation of glycogen granules and lipofuscin was evident in areas of focal myofibril lysis. Notably, only MFS patients exhibited recruitment of granulocytes, including mast cells, eosinophils and neutrophils. A subset of MFS patients displayed advanced stages of myocardial degeneration. However, these changes did not correlate with clinical myocardial failure. Conclusions To the best of our knowledge, this study represents the first evidence of ultrastructural changes in the myocardium of patients with MFS. Our data indicate various degenerative changes in the myocardial architecture, including structural and metabolic myocardial remodeling, along with inflammatory cell infiltration. We believe that our findings of ultrastructural lesions reflect the complexity of myocardial disease in MFS and emphasize the importance of careful surveillance for cardiomyopathy in MFS patients. Further studies are warranted to confirm whether the observed granulocyte influx is specific to MFS and to clarify its role in MFS myocardial disease pathophysiology.Graphical abstract

Unravelling cardiomyopathies: a comprehensive analysis of phenotypic diversity, endomyocardial biopsy patterns, and the impact of Gremlin-1 on clinical prognosis

Abstract Background Cardiomyopathies are the main cause of progressive heart failure (HF) and despite outstanding advances of diagnostics and therapies, mortality remains high.1,2 The current phenotype-based classification of non-ischemic cardiomyopathies (NICM) is based on a clinical workflow including endomyocardial biopsy (EMB).3-5 Inflammation and fibrosis are essentially involved in progression of HF and lead to development of adverse cardiovascular (CV) events.6,7 The diagnostic algorithm facilitates risk stratification of HF patients but nonetheless risk factors often remain inapparent prior to disease incidence. Objective In this study we investigate EMB patters in patients with NICM and elucidate alterations associated with phenotypic diversity and progression of HF. Methods We prospectively enrolled patients with HF due to NICM in a large-scale all-comers cohort (n=703). All patients underwent guideline-based phenotypic classification including EMB, and RNA data were acquired alongside histological analyses within the myocardium. We then performed a ten-year follow-up to screen for disease progression. Results A guideline-based classification of patients with NICM resulted in a phenotyping of aetiological risk groups (Figure 1A). We found that characteristic expression of fibroinflammatory mediators within the myocardium occurred in patients with symptomatic NICM (Figure 1B). We found that elevated expression of Gremlin-1 (Grem), a potent downstream profibrotic mediator of TGFß pathway, was associated with pro-fibrotic cardiac remodelling (Figure 1C). Further, Gremlin-1 was associated with a significant decrease of left ventricular functional capacity and inversely correlated with late gadolinium enhancement (LGE) (Figure 1D&E). Moreover, the expression of Gremlin-1 was enriched with pro-fibrotic and inflammatory RNA signalling pathways hinting at underlying pathophysiological cascades (Figure 2A&B). Most strikingly, the expression of Gremlin-1 was independently associated with an increased CV risk during the ten-year follow-up (Figure 2C). Thus, patients with Gremlin-1+ EMB were at elevated risk to suffer from all-cause mortality and number of patients eligible for ICD implantation was critically enhanced in patients with Gremlin-1+ EMB (Figure 2D-F). In addition, the estimation of an elevated CV risk by machine learning including Gremlin-1 improved the ten-year risk stratification among all patients with NICM (Figure 2G). Thus, Gremlin-1 was associated with cardiac remodelling and disease activity in HF patients (Figure 2H). Conclusion Our results unveiled that Gremlin-1 is associated with inflammation and cardiac remodelling in patients with symptomatic cardiomyopathy and patients with Gremlin-1+ EMB are at elevated risk to develop adverse CV events. Thus, the histological evaluation of Gremlin-1 may help to identify pathophysiological cascades and improve early risk discrimination and management of HF patients.Figure 1Figure 2

Early CSWT relieves myocardial fibrosis by inhibiting TGF-Smad2/3-MMP-9

Abstract Objective To investigate the correlation between the improvement of cardiac function and myocardial fibrosis and the TGF-β1-Smad2/3-MMP-9 signaling pathway in acute myocardial infarction (AMI) rats after early treatment with extracorporeal cardiac shock wave therapy (CSWT). Methods Thirty SD rats were randomly divided into five groups: sham group (6 rats), AMI group (6 rats), AMI+CSWT group (6 rats), AMI+SB431542 group (6 rats), and AMI+SB431542+CSWT group (6 rats). AMI was induced in all groups except the sham group. The AMI+CSWT group and AMI+SB431542+CSWT group received CSWT treatment. Echocardiography was performed at 4 days and 62 days postoperatively to assess left ventricular ejection fraction (LVEF) in all groups. Additionally, cardiac tissues were sampled and evaluated for pathological changes and degree of fibrosis by HE staining and Masson staining. The TGF-β1-Smad2/3-MMP-9 signaling pathway and protein expression of COL-I and COL-III were detected in the infarcted border area. Results (1) At 4 days postoperatively, EF and FS were significantly lower, and LVESV was enlarged in the AMI group, AMI+CSWT group, AMI+SB431542+CSWT group, and AMI+SB431542 group compared to the sham group (p < 0.05). (2) At 62 days postoperatively, EF and FS were significantly lower, and LVEDV and LVESV were significantly higher in the AMI group compared to the sham group (p < 0.05). EF and FS were significantly higher, and LVEDV and LVESV were significantly lower in the AMI+CSWT group compared to the AMI group (p < 0.05). There was no significant difference in EF, FS, LVEDV, and LVESV between the AMI+CSWT group and the AMI+SB431542+CSWT group (p > 0.05). (3) Histological analysis showed that the AMI group had more blue-stained areas compared to the sham group, while the AMI+CSWT group had fewer blue-stained areas and more neatly arranged red areas compared to the AMI group (p < 0.05). (4) Collagen volume fraction (CVF) was significantly higher in the AMI group compared to the sham group (p < 0.05). CVF was significantly lower in the AMI+CSWT group compared to the AMI group (p < 0.05). There was no significant difference in CVF between the AMI+CSWT group and the AMI+SB431542+CSWT group (p > 0.05). (5) The expression levels of TGF-β1, Smad3, MMP-9, COL-I, and COL-III were significantly higher in the AMI group compared to the sham group (p < 0.05). The expression levels of these proteins were significantly reduced in the AMI+CSWT group compared to the AMI group (p < 0.05). Conclusions Early CSWT improves cardiac function and inhibits myocardial fibrosis associated with the TGF-β1-Smad2/3-MMP-9 signaling pathway.pathological changesdegree of fibrosis

The role of IL15 in angiotensin II-mediated cardiac remodelling

Abstract Hypertension (HTN) mediated target organ damage, such as cardiac remodelling, is a major cause of death and disability. Cytokines are important regulators of cardiac function, inflammation, and remodelling in cardiovascular disease. Increased levels of interleukin 15 (IL15) have been noted in HTN, but its role in HTN heart disease remains unknown. This study investigates the role of the IL15 axis in the pathogenesis of cardiac remodelling in HTN. Male 12-week-old IL15 receptor α knock-out (IL15RAKO), IL15 knock-out (IL15KO) and matching wild type (WT) mice were administered vehicle or angiotensin II (AngII) (490 ng/min/kg) for 2 weeks by osmotic minipump. Blood pressure was measured by tail cuff (n=5-8). Cardiac function was analysed by echocardiography (n=3-4). Cardiac infiltrating immune cells were analysed by flow cytometry (n=2-3 for IL15RAKO, n=6-10 for IL15KO). Cardiac remodelling was assessed by picrosirius red, WGA staining (n=5-8) and expression of markers of remodelling using RT-qPCR and western blots (n=5-8). Data are presented as mean±SEM and analysed using two-way ANOVA. AngII infusion caused a significant increase in systolic blood pressure in WT and IL15KO animals. In contrast, in IL15RAKO, the response to AngII was blunted. This was in line with a decrease in ejection fraction, however only WT mice had an increase in global longitudinal strain (-20.3±2.0 vs -10.9±1.2, p=0.0039 in WT and 19.9±0.8 vs -16.0±2.1, p=0.1878 in IL15RAKO). HTN was associated with increased cardiac IL15 protein levels in mice. The heart-to-body ratio, along with cardiomyocyte cross-sectional, were significantly increased in WT, but not in IL15RAKO HTN mice. Furthermore, histological analysis showed significantly higher cardiac collagen accumulation in WT but not IL15RAKO upon AngII infusion (5.8±1.0% in WTvvs. 2.1±0.5% in IL15RAKO, p=0.0021). This was in line with a higher expression of col1a1 in WT but not IL15RAKO HTN hearts (20.0±6.0 vs 4.5±1.7, p=0.0173). Similarly, the FN1 protein level was significantly increased in WT but not IL15RAKO AngII-treated mice. These changes were associated with a higher number of Cd11b+ macrophages accumulated in WT hypertensive hearts only. In contrast to IL15RAKO, mice lacking IL15 showed similar increases in global longitudinal strain, heart hypertrophy, and cardiomyocyte cross-sectional area as WT mice upon AngII infusion. Similarly, IL15KO hypertensive hearts showed a significant increase of col1a1 and FN1 in comparison to vehicle-treated mice. A similar level of Cd11b+ macrophages was found in hypertensive IL15KO and WT mice's hearts. In conclusion, IL15RA, but not IL15, plays a critical role in cardiac remodelling mediated by AngII infusion. This suggests that the IL15-IL15RA axis may play an important yet complex role in hypertensive heart disease.

Photobiomodulation mitigates diastolic dysfunction, reduces perivascular fibrosis and inflammation, and enhances mitochondrial metabolism and angiogenesis in a mouse model of HFpEF

Abstract Introduction Emerging molecular evidence implicates mitochondrial dysfunction, systemic inflammation, and capillary rarefaction as pivotal contributors to the pathophysiology of HFpEF. Photobiomodulation (PBM) has garnered attention for its potential to augment mitochondrial metabolism, attenuate inflammation, and foster angiogenesis. This investigation aims to delineate the effects of PBM on the pathophysiology of HFpEF in a murine model. Methods To induce HFpEF, 3-month-old male C57BL/6 mice were subjected to a '2-hit model' involving a high-fat diet combined with the vasoconstrictor nitric oxide synthase inhibitor, N(G)-Nitro-L-arginine methyl ester (L-NAME). PBM therapy was administered transdermally over the cardiac region thrice weekly for a duration of 10 weeks using a 904nm super-pulsed laser, with each session lasting 90 seconds and delivering an energy density of 4.2J/cm^2 per diode to a cohort of 12 mice. A sham group of 13 mice underwent identical procedures with the laser deactivated. An additional control group of 10 mice was maintained on a standard diet without any interventions. Echocardiographic assessments were performed at the outset, 7th and 10th week time. Following the 10-week period, mice were euthanized, their hearts harvested and serum withdrawn for further analysis. Results Echocardiography of PBM-treated animals demonstrated significant attenuation of deterioration of diastolic parameters (E’) compared to the sham-treated control group (E’[mm/sec], baseline-vs-10w: PBM, -32.1±5.9-vs- -27.9±6.4; sham, -32.9±8.6-vs- -22.5±5.7; p=0.037). Histological analysis of the cardiac tissue involving picrosirius red staining for collagen revealed that the HFpEF model induced a significant increase in the standardized adventitial collagen compared to controls, which was attenuated by PBM (control 3.0±1.6; HFpEF sham: 3.9±1.8; PBM: 3.4±1.9, p=0.008 control vs sham, p=0.055 PBM vs control; p=0.396 control vs PBM). Multiplex analysis of HFpEF mice showed significantly higher levels of inflammatory chemokines than controls that were attenuated by PBM (mean±SD log[pg/ml] of the chemokine (C-X-C motif) ligand-1 (CXCL-1): PBM=2.52±0.29, Control=2.14±0.35; monocyte chemotactic protein (MCP-1): PBM=0.87±0.46, HFpEF=1.5±0.58, Control=0.8±0.6; p<0.001 and p=0.003 respectively). RNA sequencing revealed 551 genes with significant differential expression (DE) between PBM and Controls (adjusted p-value [padj]<0.1). Ingenuity Pathway Analysis (IPA) analysis revealed that PBM upregulated genes pertaining to mitochondrial metabolism and down-regulated genes active in mitochondrial dysfunction (qPCR [GAPDH] 2–∆∆Ct fold change PBM/Sham: Ndufv2=3.7, Atp5md=3.5, Dnhd1=0.5). Relevant DE genes were validated by quantitative real-time PCR (qPCR). Conclusions PBM attenuated Diastolic dysfunction, fibrosis, pro-inflammatory chemokine secretion and upregulates mitochondrial metabolism in a mouse model of HFpEF.Study designResults

Association of Neutrophil extracellular traps burden with clinical and angiographic characteristics in patients with ST-elevation myocardial infarction

Abstract Background The precise triggers for arterial plaque rupture and the underlying pathophysiology of thrombogenesis remain elusive. Polymorphonuclear neutrophils (PMN), particularly their formation of Neutrophil Extracellular Traps (NETs), have garnered attention in the context of coronary atherothrombosis. This study sought to explore the association of NETs burden with clinical and angiographic characteristics in ST-elevation myocardial infarction (STEMI) patients undergoing primary percutaneous coronary intervention (pPCI) and thrombus aspiration (TA). Methods This study included 336 consecutive STEMI patients undergoing pPCI and TA. Aspirated thrombi underwent histological analysis and NETs quantification via immunohistochemistry. Potential associations of clinical variables and angiographic outcomes with NETs burden were assessed. Results Manual TA was selectively performed in 72 cases with increased thrombotic burden and 60 thrombi were suitable for analysis. Most thrombi specimens displayed lytic features (63%) and almost three out of four specimens were identified as white thrombi. Increased NETs burden was significantly associated with prolonged pain-to-balloon time (>300 minutes) OR=10.29 (95% CI 2.11-42.22, p=0.001) and stress-induced hyperglycemia (SIH) OR=6.58 (95% CI 1.23-52.63, p=0.04) after multivariate regression analysis. Additionally, distal embolization (DE), a predictor of adverse outcomes, was more frequent among patients with an elevated NETs burden OR=16.9 (95% CI 4.23-44.52, p<0.001). Conversely, no significant association between NETs burden and final TIMI flow=3 was observed. Conclusion Elevated NETs burden in STEMI thrombi is linked to delayed reperfusion, SIH and increased risk of DE. Further research is needed to elucidate the role of NETs as a potential therapeutic target in acute atherothrombosis.

Alveolar Ridge Regeneration With Open Versus Closed Healing in Damaged Extraction Sockets: A Preclinical InVivo Study.

The objective of this study was to compare open versus closed healing of soft and hard tissue following alveolar ridge preservation (ARP) procedures in damaged extraction sockets. ARP was performed in five mongrel dogs using collagenated deproteinized bovine bone mineral (cDBBM) and a resorbable non-cross-linked collagen membrane (NCCM) in damaged extraction sockets, with each socket entrance left either open (open group) or closed (closed group). Clinical wound epithelization at the socket entrance and the dimensions of keratinized tissue were evaluated over time. Additionally, the augmented ridge dimensions and new bone formation were assessed radiographically and histologically at 8 weeks after surgery. The dimensions of the socket entrance gradually decreased in the open group, and wound epithelization was almost complete within 4 weeks. The mucogingival junction was maintained more apically in the open group than in the closed group (0.14 ± 0.40 mm vs. -0.86 ± 0.71 mm [mean ± SD], p < 0.05). The augmented ridge dimensions did not differ significantly between the open and closed groups (93.1% ± 5.4% vs. 88.3% ± 11.2%, p > 0.05). Histological analyses revealed no significant differences in the amount of newly formed bone. However, membrane resorption in the crestal region was more pronounced in the open group. Open and closed healing approaches for ARP in extraction sockets with damaged buccal wall resulted in similar ridge dimensions and new bone formation. However, there was less reduction of the buccal bone crest and wider keratinized tissue width after open healing.

Decomposition and Changes in InVivo Post-HA Filler Injection: A Review.

Hyaluronic acid (HA) fillers are widely used in aesthetic medicine, but their invivo behavior and long-term effects are not fully understood. To review the decomposition and changes occurring in the body following HA filler injections, focusing on crosslinking agents, degradation processes, and tissue responses. This review analyzed oxidative and enzymatic degradation processes of HA fillers, evaluated the impact of 1,4-Butanediol Diglycidyl Ether (BDDE) crosslinking, and examined histological changes post-injection. Uncrosslinked HA degrades rapidly due to endogenous hyaluronidase, while crosslinked HA undergoes slower degradation via free radicals and hyaluronidase. Complete cross-linking (C-MoD) showed better durability compared to partially cross-linked BDDE (P-MoD). The concept of modification efficiency (MoE) was proposed to optimize filler safety and viscoelastic properties. Histological analysis revealed collagen capsule formation and autologous tissue replacement, affecting long-term outcomes. The degree of chemical modification (MoD) influences filler durability and safety, with concerns raised about potential delayed immune reactions from accumulated pendent BDDE. Clinicians should consider injection site, tissue conditions, and filler properties for safe and effective HA filler use. Emphasizing thorough BDDE removal and optimal crosslinking can enhance treatment safety and efficacy. The balance between achieving desired viscoelastic properties and minimizing potential risks is crucial. Future studies should include diverse ethnic groups to validate findings and further explore long-term tissue responses to HA fillers.

Histological ovarian features and hormonal determinations in assigned females at birth transgender individuals according to different testosterone preparations

IntroductionDistinct androgen formulations have been used as gender-affirming hormone treatment, but little is known about the specific changes that may occur in the ovary depending on the testosterone preparation used. The study aims to evaluate the histological modifications of the ovarian tissue and the hormonal changes after gender-affirming surgery based on the testosterone preparation employed, such as testosterone cypionate or undecanoate.DesignUnicenter transversal cohort study.Materials and methodsSixty transmasculine persons before and after gender-affirming surgery. A histological examination of the ovaries was conducted, including the follicular population and the characterization of the ovarian stroma. Hormonal status (testosterone, estradiol, FSH, and LH) were also assessed before and after the procedure.ResultsThe median age of participants was similar between the two groups (27.9 vs. 26.7 years, p = 0.27). There were no differences in all hormonal determinations before gender-affirming surgery between the groups. After surgery, FSH levels increased significantly, especially in the testosterone undecanoate group compared to the cypionate group (72.3 vs. 38.3 U/L, p = 0.02), consistent with LH determinations (43.0 vs. 23.4 U/L, p = 0.02). However, no regimen modification was required for any individual. No statistical differences were observed in any parameter concerning the follicular population, nor were there any variances in the thickness of the tunica albuginea (p = 0.85) or the proportion of luteinized stromal cells. Nevertheless, there was a tendency toward increased luteinization in the testosterone cypionate group (88.2% vs. 76.9%, p &amp;gt; 0.05).ConclusionsIn a cohort of transmasculine individuals using different androgen preparations, histological analysis of ovarian tissue revealed comparable findings. Both groups exhibited similar follicular populations and comparable modifications in stromal tissue. However, significant differences were observed in hormonal profiles, although no modification in testosterone dosage was needed.

Stress memory of heart failure in hematopoietic niche promotes recurrence via adipocytic skewing of mesenchymal stromal cells

Abstract Background Heart failure (HF) is marked by recurrent acute decompensations, which poses significant treatment challenges in advanced stages. Our preceding work demonstrated that hematopoietic stem cells (HSCs) from HF-experienced mice induce cardiac fibrosis and dysfunction when transplanted, as a result of epigenetic alterations favoring myeloid hematopoiesis and impeding the differentiation of monocytes into cardiac mature macrophages. These findings hint at epigenetic modifications in HSCs as potential drivers of HF recurrence, suggesting their manipulation as a novel therapeutic strategy. However, the exact mechanisms by which HF-associated stress leads to these epigenetic changes remain unclear. Given the crucial role of bone marrow mesenchymal stromal cells (MSCs) in maintaining HSC stemness, this study explores the impact of cardiac stress on MSC function and its subsequent effect on HSCs. Purpose This study aims to elucidate the mechanisms behind the phenotypic changes in HSCs and to identify novel therapeutic targets to mitigate HF recurrence by investigating the role of MSCs in the hematopoietic niche under cardiac stress. Methods &amp; Results Initially, we examined MSC phenotypic changes during HF using bulk and single-cell RNA sequencing. Results from transverse aortic constriction (TAC) model mice revealed a shift towards adipocyte differentiation under cardiac stress. Histological analyses confirmed an increase in adipocytes in TAC mice compared to controls, suggesting a stress-induced predisposition of MSCs towards adipocytic lineage commitment. This trend was further supported by ex vivo models using MSCs isolated from TAC mice. Notably, the proportion of adipocyte lineage-specific MSCs correlated with cardiac dysfunction severity. Subsequently, we assessed the impact of HF-conditioned MSCs on HF development by transplanting healthy HSCs with MSCs from control or TAC mice. Mice receiving HF-conditioned MSCs developed HF, evidenced by reduced ejection fraction and cardiac fibrosis. In these mice, a notable increase in HSC proliferation and a relative increase of myeloid cells within the peripheral blood were observed, along with an increase in proinflammatory cardiac macrophages. These findings suggest HF induces a "stress memory" in bone marrow MSCs. To test the potential of inhibiting adipocyte differentiation of MSCs in erasing this stress memory, we administered MSC phenotype-modulating agents to TAC mice, resulting in improved cardiac function and the disappearance of adipocytes from the bone marrow. Conclusions This study sheds light on how cardiac stress affects the bone marrow niche, leading to adipocytic skewing of MSCs and subsequent alterations in HSCs, thereby exacerbating cardiac dysfunction. The potential of targeting bone marrow niche components in HF treatment is highlighted.

Beneficial effects of the SGLT2 inhibition on oxidative skeletal muscle in infarcted rats

Abstract Introduction Metabolic, morphological, and biochemical changes in skeletal muscle are common in chronic heart failure patients. Despite the cardioprotective effects of sodium-glucose co-transporter 2 (SGLT2) inhibition in heart failure, the impact of this drug on skeletal muscle remains poorly understood. This study investigated the effects of SGLT2 inhibitor empagliflozin (EMPA) on the soleus muscle of rats with myocardial infarction (MI)-induced heart failure. Methods One week after MI induction, male Wistar rats were divided into Sham (n=10), Sham+EMPA (n=12), MI (n=10) and MI+EMPA (n=09) groups. EMPA was added to rat chow (5 mg/kg/day) for 12 weeks. Cardiac remodeling was evaluated by echocardiogram. Enzymatic activity and oxidative stress marker concentrations were assessed by spectrophotometry; protein expression was evaluated by Western blotting. After histological analysis of the left ventricle (LV), only rats with MI greater than 35% of total LV area were included in the study. Statistical analysis: ANOVA and Tukey or Kruskal–Wallis and Dunn and t test; p&amp;lt;0.05. Results Infarction size did not differ between groups. Infarcted groups had larger LV systolic and diastolic diameters, LV diastolic area (Sham 47±7.1; Sham+EMPA 48±4.5; MI 100±16.1*; MI+EMPA 85±10.1#† mm2; p&amp;lt;0.05: * vs Sham; # vs Sham+EMPA; † vs MI), and left atrial diameter (Sham 5.62±0.2; Sham+EMPA 5.65±0.3; MI 7.69±1.3*; MI+EMPA 6.85±0.9#† mm; p&amp;lt;0.05: * vs Sham; # vs Sham+EMPA; † vs MI); and lower LV posterior wall shortening velocity and ejection fraction than control groups. Echocardiographic changes were attenuated in MI+EMPA compared to MI. Other results are shown in Picture 1. Conclusion SGLT2 inhibitor empagliflozin attenuates infarction-induced cardiac remodeling in rats. Empagliflozin prevents increase in oxidative stress, modulates protein turnover by IGF-1 pathway, and attenuates morphological and biochemical changes in the soleus muscle of infarcted rats.

Anti-VEGFR2 neutralising antibody slows the progression of multistep oral carcinogenesis.

Angiogenesis plays an important role in cancer growth and metastasis, and it is considered a therapeutic target to control tumour growth following anti-angiogenic therapy. However, it is still unclear when tissues initiate angiogenesis during malignant transformation from premalignant condition and whether this premalignant condition could be a therapeutic target of anti-angiogenic therapy. In this study, we aimed to analyse the onset of angiogenesis by evaluating morphological and functional alterations of microvessels during oral multistep carcinogenesis using a 4-nitroquinoline 1-oxide (4NQO)-induced oral carcinogenesis mouse model. In the study, we initially confirmed that with the use of 4NQO, oral lesions develop in a stepwise manner from normal mucosa through oral epithelial dysplasia (OED) to oral squamous cell carcinoma (OSCC). Evaluation of CD31-immunostained specimens revealed that microvessel density (MVD) increases in a stepwise manner from OEDs. Histological and functional analyses revealed the structural abnormalities and leakage of blood vessels had already taken place in OED. Then we evaluated the expression profiles of Hif1a and Vegfa along with hypoxic status and found that OED exhibited increased Vegfa expression under hypoxic conditions. Finally, we tested the possibility of OEDs as a target of anti-angiogenic therapy and found that anti-VEGFR2 neutralising antibody in OED slowed the disease progression from OED to OSCC. These data indicate that an angiogenic switch occurs at the premalignant stage and morphological, and functional alterations of microvessels already exist in OED. These findings also elucidate the tumour microenvironment, which gradually develops along with carcinogenic processes, and highlight usefulness of the 4NQO-induced carcinogenesis model in the study of epithelial and stromal components, which will support epithelial carcinogenesis. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

The Role of CLDN18.2 in Gastric Cancer Prognosis: A Systematic Review and Meta-Analysis

Background Gastric cancer (GC) is a major global cause of cancer mortality, with a median overall survival of just 12 months. CLDN18.2, a specific isoform of Claudin18 normally expressed in the gastric mucosa, has emerged as a potential therapeutic target and prognostic biomarker due to its exposure on the surface of tumor cells following malignant transformation. This exposure allows CLDN18.2's extracellular loops to bind monoclonal antibodies, presenting new opportunities for targeted therapy and improved prognostic assessment. Methods A comprehensive search of PubMed, EMBASE, Cochrane Library and Web of Science databases was conducted for studies that addressed the correlation of CLDN18.2 with: (1) Progression-free survival (PFS), and (2) Overall Survival (OS). Hazard ratio (HR) and odds ratio (OR) with 95% confidence intervals (CIs) were calculated using a fixed-effects model. Heterogeneity was examined with I2 statistics. P values of ≤ 0.05 were considered statistically significant. Statistical analyses were performed using RStudio, version 4.2.3. Results A total of 15 studies encompassing a total of 4,085 patients were included. There were 2,691 (65.8%) male, and 1,394 (34.2%) female patients. In the histologic GC analysis, there were 1,582 (38.7%) patients that had intestinal type and 1,280 (31.3%) with diffuse type. Patients with CLDN18.2 negative status exhibited a non-significant trend towards prolonged PFS (HR: 1.25; 95% CI: 0.98-1.61; p = 0.07; I2=18%) and a significant prolonged OS (HR: 1.20; 95% CI: 1.07-1.34; p < 0.01; I2=37%) when compared to CLDN18.2-positive patients. Conclusion Our findings establish CLDN18.2 as a robust negative prognostic indicator for overall survival in GC patients. While its impact on PFS was not statistically significant, the association with OS suggests CLDN18.2 may serve as a marker for complex biological processes underlying tumor advancement.

A novel spine tester TO GO.

Often after large animal experiments in spinal research, the question arises-histology or biomechanics? While biomechanics are essential for informed decisions on the functionality of the therapy being studied, scientists often choose histological analysis alone. For biomechanical testing, for example, flexibility, specimens must be shipped to institutions with special testing equipment, as spine testers are complex and immobile. The specimens must usually be shipped frozen, and, thus, biological and histological investigations are not possible anymore. To allow both biomechanical and biological investigations with the same specimen and, thus, to reduce the number of required animals, the aim of the study was to develop a spine tester that can be shipped worldwide to test on-site. The "Spine Tester TO GO" was designed consisting of a frame with three motors that initiate pure moments and rotate the specimen in three motion planes. A load cell and an optical motion tracking system controlled the applied loads and measured range of motion (ROM) and neutral zone (NZ). As a proof of concept, the new machine was validated and compared under real experimental conditions with an existing testing machine already validated employing fresh bovine tail discs CY34 (n = 10). The new spine tester measured reasonable ROM and NZ from hysteresis curves, and the ROM of the two testing machines formed a high coefficient of determination R 2 = 0.986. However, higher ROM results of the new testing machine might be explained by the lower friction of the air bearings, which allowed more translational motion. The spine tester TO GO now opens up new opportunities for on-site flexibility tests and contributes hereby to the 3R principle by limiting the number of experimental animals needed to obtain full characterization of spine units at the macroscopic, biomechanical, biochemical, and histological level.