Fibrotic Areas Research Articles

Empagliflozin alleviates diabetic cardiomyopathy via inhibiting excessive autophagy and adverse remodeling in type 1 diabetic mice

Abstract Sodium-glucose cotransporter 2 (SGLT2) inhibitors reduce cardiovascular mortality in patients with and without diabetes mellitus, but underlying mechanisms remain unclear. Autophagy is an important cardioprotective mechanism under acute stress conditions, but excessive autophagy can accelerate myocardial cell death leading to autosis. In this study we evaluated the role of empagliflozin (EMPA) against cardiac injury in streptozotocin (STZ)-induced diabetic mice, and assessed underlying mechanisms. EMPA (10 mg/kg/die)-treated diabetic male mice had a lower number of TUNEL-positive apoptotic cells (%TdT+ 4.9±2, p<0.05), lower percentage of SA-β-gal positive senescent cardiac area (%SA-β-gal 7.9±1, p<0.05), lower fibrotic area (%picrosirius red 0.15±0.06, p<0.05), lower autophagy (%green fluorescence 2.3±0.6, p<0.01), compared to diabetic mice (%TdT+ 4.9±2; %SA-β-gal 10±2; %picrosirius red fibrotic area 0.24±0.08; %green fluorescence autophagic area 3.98±0.5). Proteomics analysis of cardiac tissue showed downregulation of apoptosis, necrosis and 5' adenosine monophosphate-activated protein kinase (AMPK) pathways, along with upstream activation of vascular endothelial growth factor and sirtuins in the heart of EMPA+STZ treated mice compared to STZ mice. Since activation of sirtuins leads to modulation of cardiomyogenic transcription factors, we analyzed total expression of serum response factor (SRF), myocardin and myocardin-related transcription factor (MRTF), and DNA binding activity of SRF by electromobility shift assay. While EMPA only increased MRTF, EMPA (2.2±0.01, p<0.01) and EMPA+STZ (2.0±0.1, p<0.01) significantly increased SRF/SRE binding activity compared with STZ alone (0.5±0.01), which was paralleled by the increase in cardiac actin expression (4.06±0.1 vs 2.2±0.01, p<0.01). EMPA significantly reversed STZ-induced systolic cardiac dysfunction at echocardiography, and downregulation of pS368-connexin 43 active isoform (Table). In parallel in vitro experiments, EMPA inhibited excessive autophagy in high glucose-treated cardiomyocytes by inhibiting the autophagy inducer GSK3β, leading to reactivation of transcriptional complex for cardiomyocyte contractile genes. Our results indicate that EMPA plays a cardioprotective effect by inhibiting excessive autophagy through the mTOR/AMPK axis in the mouse model of STZ-induced type 1 diabetes. EMPA also promotes cardiac reverse remodeling by inhibiting fibrosis, activates cardiac Cx43 and the promyogenic transcriptional program through the sirtuins-SRF axis (Figure 1), in the context of diabetes. Funding Acknowledgement Type of funding sources: None.

Myocardial inflammation in ischaemic and non-ischaemic heart failure relates to impaired mitochondria

Abstract Background Mitochondrial dysfunction is a driving factor in the development of heart failure (HF) and relates to poor cardiac function. Through elevated oxidative stress it is linked to myocardial inflammation. Both mechanisms promote the development of cardiac fibrosis, a key contributor to adverse outcomes in ischaemic and non-ischaemic HF. Differences in myocardial mitochondrial function and inflammation between patients with ischaemic and non-ischaemic HF have been indicated. While immunosuppression in non-ischaemic HF with myocardial inflammation has improved long-term results, little is known about possible benefits in ischaemic HF. Purpose We hypothesised that: 1. Myocardial lymphocytic inflammation and fibrosis differ in patients with HF due to ischaemic cardiomyopathy (ICM) compared to patients with dilated cardiomyopathy (DCM). 2. Inflammation and fibrosis are associated with markers of mitochondrial dysfunction. Methods Myocardial tissue specimens were obtained from the left ventricular (LV) apex of 65 patients (n[ICM] = 33, n[DCM] = 32) with HF requiring an LV assist device or heart transplantation. We assessed oxidative phosphorylation capacity (OXPHOS) via high resolution respirometry in saponine-permeabilised myocardial fibres (OROBOROS Oxygraph-2k). Reactive oxygen species (ROS) production was measured fluoroscopically via the Amplex Red method. Immunohistochemistry staining for CD3 was performed on snap-frozen tissue and digitally analysed. Azan staining was used for quantification of fibrotic area. Statistical analysis was conducted with GraphPad Prism v9.0 and IBM SPSS v27.0. Results ICM and DCM patients did not differ significantly regarding age (60.9±5.4 vs. 55.8±12.4 years, p=0.11), sex (91% vs. 90% male, p=0.93), BMI (27.6±5.1 vs. 26.5±6.0 kg/m2, p=0.82), or diabetic status (31% vs. 20% Type 2 Diabetes mellitus, p=0.31). Tissue specimens of ICM patients displayed about three times larger fibrotic areas compared to DCM (20.9±21.2 vs. 7.2±5.6%, p=0.001). Cellular inflammation (>14 CD3+ cells/mm2) was significantly more common in ICM than DCM patients. (27 vs. 6%, p=0.024). Inflammation and fibrosis did not correlate significantly (r=0.09, p=0.46). Fibrosis was not associated with OXPHOS capacity (r=−0.129, p=0.33). In patients with inflammation, OXPHOS capacity was lower compared to patients without inflammation (92.3±40.1 vs. 131.6±55.6 pmol/(s*mg), p=0.031) and inflammation was associated with lower OXPHOS capacity (r=−0.296, p=0.019). There was a trend towards lower electron transfer capacity in patients with inflammation (98.6±43.1 vs. 129.7±48.3 pmol/(s*mg), p=0.053). ROS production was not significantly different between tissue specimens with or without inflammation (0.51±0.21 vs. 0.67±0.33 pmol/(s*ml), p=0.17). Conclusions Myocardial cellular inflammation is prominent in ICM patients and relates to impaired mitochondria. Future studies may evaluate possible benefits from immunosuppression in ICM patients. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): German Research Foundation (DFG)

Regulation of cardiomyocyte adhesion and mechanosignalling through distinct nanoscale behaviour of integrin ligands mimicking healthy or fibrotic extracellular matrix.

The stiffness of the cardiovascular environment changes during ageing and in disease and contributes to disease incidence and progression. Changing collagen expression and cross-linking regulate the rigidity of the cardiac extracellular matrix (ECM). Additionally, basal lamina glycoproteins, especially laminin and fibronectin regulate cardiomyocyte adhesion formation, mechanics and mechanosignalling. Laminin is abundant in the healthy heart, but fibronectin is increasingly expressed in the fibrotic heart. ECM receptors are co-regulated with the changing ECM. Owing to differences in integrin dynamics, clustering and downstream adhesion formation this is expected to ultimately influence cardiomyocyte mechanosignalling; however, details remain elusive. Here, we sought to investigate how different cardiomyocyte integrin/ligand combinations affect adhesion formation, traction forces and mechanosignalling, using a combination of uniformly coated surfaces with defined stiffness, polydimethylsiloxane nanopillars, micropatterning and specifically designed bionanoarrays for precise ligand presentation. Thereby we found that the adhesion nanoscale organization, signalling and traction force generation of neonatal rat cardiomyocytes (which express both laminin and fibronectin binding integrins) are strongly dependent on the integrin/ligand combination. Together our data indicate that the presence of fibronectin in combination with the enhanced stiffness in fibrotic areas will strongly impact on the cardiomyocyte behaviour and influence disease progression.This article is part of the theme issue ‘The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease’.

MRI-based numerical modeling of cardiac pulsed field ablation (PFA) with sub-microsecond pulses

Abstract Background The use of irreversible electroporation (IRE) for cardiac ablation in the management of cardiac arrhythmias, known as Pulsed Field Ablation (PFA), has gained the attention of many researchers, physicians and companies. Despite the demonstrated efficacy and safety profiles of the technique, there are still many open questions. One of them deals with the effective electric field thresholds necessary for cardiac tissue ablation. Contrary to radiofrequency (RF) ablation, where the thermotemporal thresholds for tissue damage are well established, the electric field thresholds for effective IRE are uncertain and depend on the tissue type and the waveform characteristics of the delivered electric field. Purpose The use of numerical models to understand the electric field distribution and the corresponding lesion for a specific electrode geometry are widely used in IRE for cancer treatment. Models can predict ablation volumes when the field thresholds are known or can be used to estimate the field thresholds when the lesion geometry is established post-treatment. The goal of the present study was to build a subject-specific realistic 3D numerical model to assess the ability of modeling to predict PFA lesions. Methods PFA experiments were performed in male mongrel dogs following a protocol approved by the Mayo Clinic Institutional Animal Care and Use Committee. Briefly, animals were sedated and intubated, and two non-irrigated deflectable catheters with an 8-mm tip were positioned on both sides of the interventricular septum under fluoroscopy guidance. Submicrosecond pulses were delivered in a bipolar fashion across both catheters. Sequences of pulses of 300 ns duration (or other durations in that range) were repetitively applied with voltage levels in the kV range. 30 days after the procedure, MRI with late gadolinium enhancement (LGE) were acquired to assess the presence of ablation lesions in the treated tissue. Ventricular areas were segmented from MRI images and 3D volumes were created. The generated 3D geometries were imported into software COMSOL Multiphysics. Realistic 3D geometries of the catheters were placed in the same positions than during the experiments, fluoroscopy videos recorded during the procedures were used as a guidance. The computed electric field intensity distributions were compared to the LGE areas to extract the range of electric field thresholds where the predicted lesion size was compatible with the real size of the LGE areas for each application site and pulsing protocol. Results and conclusions Our results show how the numerical model is able to reproduce the shape of the LGE fibrotic lesion areas observed 30 days after the procedure. Additionally, the model clearly confirms the dose-dependent lesion sizes observed experimentally. This study supports that numerical modeling is a valuable tool for understanding PFA treatments and that it could be used as patient-specific treatment planning tool. Funding Acknowledgement Type of funding sources: Public grant(s) – EU funding. Main funding source(s): European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie

Activation of cardiac macrophages, endothelial cells and fibroblasts in experimental autoimmune myocarditis

Abstract Background/Introduction Inflammation of cardiac tissue, termed myocarditis, is a common cause of dilated cardiomyopathy associated with abnormal tissue remodeling, stiffening of ventricles and heart failure. Experimental autoimmune myocarditis (EAM) represents CD4+ T cell-dependent animal model of acute myocarditis followed by development of cardiomyopathy, cardiac fibrosis and systolic dysfunction. Purpose The aim of the study was to investigate the role of cardiac macrophages, endothelial cells and fibroblasts in myocarditis and post-inflammatory cardiomyopathy in mouse model of EAM. Methods EAM was induced in BALB/c mice by immunization with alpha myosin heavy chain and complete Freund's adjuvant. Reporter mice expressing EGFP under collagen type I promoter (Coll-EGFP), transgenic αSMA-TK mice with ganciclovir-inducible ablation of proliferating myofibroblasts and Rosa26-YFP/LysM-Cre and Rosa26-YFP/LysM-Cre/Tgfbr2 fl/fl with YFP expression and Tgfbr2 deletion in myeloid cell population were used in this study. Cardiac fibroblasts and macrophages were sorted using BD FACSAria™ II Cell Sorter and analyzed for the genome transcriptomics by RNA sequencing. Echocardiography was performed on Vevo 2100 Imaging System. Cardiac fibrosis was measured as percentage of fibrotic area using Trichrome Massons's staining and by hydroxyproline assay. Cardiac hypertrophy was analyzed as means of cross-sectional cardiomyocyte area. Monocytes and endobcells were analyzed using BD FACSCanto™ II flow cytometry. Results Cardiac fibroblasts in response to acute myocarditis (d21 of EAM) showed activation of immune processes (mainly chemokine production such as Ccl6, Ccl9, Cxcl2, Cxcl3, Cxcl5, Cxcl9, Cxcl13), cytoskeletal re-organization (Cxadr, F11r, Gdpd2, Krt8, Krt19, Ptk2b, Rac2, Rhov, Rnd1, S100a9, Spire2, Was) and upregulation of genes involved in extracellular matrix turnover (Bmp7, Kng2, Lgals3, Cthrc1, Cela1, Spn) including collagens. Ablation of myofibroblasts (between d21–40 of EAM) resulted in markedly reduced heart weight and cardiomyocyte hypertrophy, attenuated expression of genes related to hypertrophy (Acta1, Actc1, Bnp, Cfl2, Pdlim5), improved stroke volume, ejection fraction and cardiac output but did not prevent development of post-inflammatory cardiac fibrosis measured at d40 of EAM. Analysis of monocytes and endothelial cells indicated excessive production of type I collagen by these cells at d21. Analysis of cardiac macrophages pointed out TGF-β-dependent expression of cytokines (Ifn, Il23a, Il10, Il12b, Cxcl1, Tnf) and theirs receptors (Cxcr1, Ccr4) at d21 of EAM. Conclusions Acute myocarditis activates proinflammatory and profibrotic responses in cardiac resident cells. Our data suggest that cardiac myofibroblasts play a particularly important role in development of post-inflammatory cardiomyopathy and heart failure. Targeting cardiac myofibroblasts might therefore represent a novel therapeutic strategy in inflammatory heart disease. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Science Centre (Poland)

Hematogenous Macrophages Contribute to Fibrotic Scar Formation After Optic Nerve Crush.

Although glial scar formation has been extensively studied after optic nerve injury, the existence and characteristics of traumatic optic nerve fibrotic scar formation have not been previously characterized. Recent evidence suggests infiltrating macrophages are involved in pathological processes after optic nerve crush (ONC), but their role in fibrotic scar formation is unknown. Using wild-type and transgenic mouse models with optic nerve crush injury, we show that macrophages infiltrate and associate with fibroblasts in the traumatic optic nerve lesion fibrotic scar. We dissected the role of hematogenous and resident macrophages, labeled with Dil liposomes intravenously administered, and observed that hematogenous macrophages (Dil+ cells) specifically accumulate in the center of traumatic fibrotic scar while Iba-1+ cells reside predominantly at the margins of optic nerve fibrotic scar. Depletion of hematogenous macrophages results in reduced fibroblast density and decreased extracellular matrix deposition within the fibrotic scar area following ONC. However, retinal ganglion cell degeneration and function loss after optic nerve crush remain unaffected after hematogenous macrophage depletion. We present new and previously not characterized evidence that hematogenous macrophages are selectively recruited into the fibrotic core of the optic nerve crush site and critical for this fibrotic scar formation.

HIPK2 attenuates bleomycin-induced pulmonary fibrosis by suppressing the Wnt/β-catenin signaling pathway.

The present study aimed to investigate the effect of homeodomain interacting protein kinase 2 (HIPK2) on pulmonary fibrosis and the probable mechanisms. We constructed a mouse model of bleomycin-induced pulmonary fibrosis and up-regulated the expression of HIPK2 in the lung by in vivo transfection. Lung tissues were collected for the detection of mesenchymal markers (α-SMA, collagen I, collagen III) and the expression of β-catenin as assessed by RT-PCR, western blot, and immunohistochemistry. Mouse lung fibroblasts (MLFs) with upregulation or downregulation of HIPK2 were successfully constructed and XAV939 was used to downregulate β-catenin expression. Then, we evaluated the activation of MLFs and the Wnt/β-catenin pathway under various conditions. The results showed that in the bleomycin-induced mouse model group, the lung alveolar structure was severely damaged, the amount of collagen fibers was increased in alveolar speta, and the expression of HIPK2 in the fibrotic area was found to be reduced. After upregulating HIPK2 in the lungs of the mouse fibrosis model we found that pulmonary fibrosis was attenuated and the expression of β-catenin and mesenchymal markers was reduced. The upregulation of HIPK2 inhibited the proliferation and migration of MLFs induced by TGF-β1, promoted apoptosis of MLFs, and reduced the expression of mesenchymal markers and β-catenin. Meanwhile, downregulation of HIPK2 promoted the proliferation and migration of MLFs, inhibited apoptosis, and promoted mesenchymal markers and β-catenin expression. XAV939 treatment of MLFs silencing HIPK2 inhibited their proliferation and activation via silencing HIPK2, promoted apoptosis, and reduced interstitial markers and β-catenin expression. HIPK2 can attenuate bleomycin-induced pulmonary fibrosis by inhibiting the Wnt/β-catenin pathway in mouse lung fibroblasts.

Atrial fibrosis identification with unipolar electrogram eigenvalue distribution analysis in multi-electrode arrays

Atrial fibrosis plays a key role in the initiation and progression of atrial fibrillation (AF). Atrial fibrosis is typically identified by a peak-to-peak amplitude of bipolar electrograms (b-EGMs) lower than 0.5 mV, which may be considered as ablation targets. Nevertheless, this approach disregards signal spatiotemporal information and b-EGM sensitivity to catheter orientation. To overcome these limitations, we propose the dominant-to-remaining eigenvalue dominance ratio (EIGDR) of unipolar electrograms (u-EGMs) within neighbor electrode cliques as a waveform dispersion measure, hypothesizing that it is correlated with the presence of fibrosis. A simulated 2D tissue with a fibrosis patch was used for validation. We computed EIGDR maps from both original and time-aligned u-EGMs, denoted as mathcal {R} and mathcal{R}^{mathcal{A}}, respectively, also mapping the gain in eigenvalue concentration obtained by the alignment, Delta mathcal{R}^{mathcal{A}}. The performance of each map in detecting fibrosis was evaluated in scenarios including noise and variable electrode-tissue distance. Best results were achieved by mathcal{R}^{mathcal{A}}, reaching 94% detection accuracy, versus the 86% of b-EGMs voltage maps. The proposed strategy was also tested in real u-EGMs from fibrotic and non-fibrotic areas over 3D electroanatomical maps, supporting the ability of the EIGDRs as fibrosis markers, encouraging further studies to confirm their translation to clinical settings.Graphical Upper panels: map of mathcal {R}^{mathcal {A}} from 3×3 cliques for Ψ= 0∘ and bipolar voltage map Vb-m, performed assuming a variable electrode-to-tissue distance and noisy u-EGMs (noise level σv = 46.4 μV ). Lower panels: detected fibrotic areas (brown), using the thresholds that maximize detection accuracy of each map

Artificial intelligence based liver portal tract region identification and quantification with transplant biopsy whole-slide images

Liver fibrosis staging is clinically important for liver disease progression prediction. As the portal tract fibrotic quantity and size in a liver biopsy correlate with the fibrosis stage, an accurate analysis of portal tract regions is clinically critical. Manual annotations of portal tract regions, however, are time-consuming and subject to large inter- and intra-observer variability. To address such a challenge, we develop a Multiple Up-sampling and Spatial Attention guided UNet model (MUSA-UNet) to segment liver portal tract regions in whole-slide images of liver tissue slides. To enhance the segmentation performance, we propose to use depth-wise separable convolution, the spatial attention mechanism, the residual connection, and multiple up-sampling paths in the developed model. This study includes 53 histopathology whole slide images from patients who received liver transplantation. In total, 6,012 patches derived from 30 images are used for our deep learning model training and validation. The remaining 23 whole slide images are utilized for the model testing. The average liver portal tract segmentation performance of the developed MUSA-UNet is 0.94 (Precision), 0.85 (Recall), 0.89 (F1 Score), 0.89 (Accuracy), 0.80 (Jaccard Index), and 0.91 (Fowlkes–Mallows Index), respectively. The clinical Scheuer fibrosis stage presents a strong correlation with the resulting average portal tract fibrotic area (R = 0.681, p<0.001) and portal tract percentage (R = 0.335, p = 0.02) computed from the MUSA-UNet segmentation results. In conclusion, our developed deep learning model MUSA-UNet can accurately segment portal tract regions from whole-slide images of liver tissue biopsies, presenting its promising potential to assist liver disease diagnosis in a computational manner.

Abstract 090: Cells Of The Renin Lineage Promote Kidney Regeneration Post - Release Of Ureteral Obstruction In Neonatal Mice.

Renin, the rate-limiting enzyme of the renin-angiotensin system, is increased in the kidneys subjected to ureteral obstruction. We elucidated the changes and contribution of renin-producing cells (RPCs) and the cells of renin lineage (CoRL) during kidney damage and regeneration using a partial unilateral ureteral obstruction (pUUO) model in neonatal mice. Renin cells were ablated by cell-specific expression of diphtheria toxin A chain (DTA) and labeled with GFP. We compared the cell fate changes between kidneys from the wild-type (WT) and the DTA-expressing animals, using the pUUO model. The kidneys of 3W old DTA-positive Ren1 dDTA/+ ;R26R DTA/+ and Ren1 dCre/+ ;R26R DTA/+ genotypes were grossly normal. We observed no significant differences in the Myh11-positive renal vascular area between the WT and the DTA-positive mice (WT-3W: 1.72±0.10 % (n=6), Ren1 dDTA/+ ;R26R DTA/+ -3W: 1.94 ±0.123 % (n=7), Ren1 dCre/+ ;R26R DTA/+ -3W: 2.017±0.16% (n=4)). In the kidneys of the WT group, RPCs and the CoRL significantly increased at 3W post-obstruction and the distribution was similar to the sham-operated kidneys at 2W post-relief of obstruction (Renin-positive area-WT: Sham-3W: 0.57±0.02% (n=6), Obstructed-3W: 1.59±0.44% (n=4), Post-release-2W: 0.71±0.12% (n=3)). There was a significant decrease in RPCs in the DTA animals, and the reduction of RPCs and CoRL was markedly higher in Ren1 dCre/+ ;R26R DTA/+ mice in comparison to Ren1 dDTA/+ ;R26R DTA/+ group. Also, the DTA-positive animals did not respond to pUUO with a significant increase in renin as in the WT (Renin-positive area: Ren1 dDTA/+ ;R26R DTA/+ Sham-3W: 0.13±0.048% (n=5), Obstructed-3W: 0.23±0.10% (n=5), Post-release-2W: 0.14±0.04% (n=9) Ren1 dCre/+ ;R26R DTA/+ Sham: 0.010±0.004% (n=4), Obstructed: 0.07±0.004% (n=4), Post-release: 0.03±0.004% (n=3)). The renal fibrotic area and Tgfb1 mRNA levels post-obstruction and relief of obstruction were significantly high in Ren1 dCre/+ ;R26R DTA/+ group in comparison to Ren1 dDTA/+ ;R26R DTA/+ genotype due to the depletion of CoRL. Thus, our current study reveals that CoRL undergoes cell fate changes in response to obstruction and the release of obstruction, and these changes play a critical role in promoting kidney regeneration post-relief of obstruction.

Cerebellar fastigial nucleus electrostimulation attenuates inflammation in a Post-Infarction rat model by activating cholinergic anti-inflammatory pathway

There are negative correlations between indices of heart rate variability (HRV) and markers of inflammation. The inflammation plays an important role in myocardial damages after myocardial infarction (MI). Our previous study found that fastigial nucleus electrostimulation (FNS) improved abnormal HRV in a rat model of MI. Whether it can reduce inflammation and improve cardiac function after MI and the underlying mechanisms remain unknown. 66 Sprague Dawley rats were randomly divided into 4 groups as follows: i) Sham group (sham operation); ii) MI group (left anterior descending coronary artery ligation); iii) FNS + MI group (left fastigial nucleus electrostimulation plus MI); iv) FNL + FNS + MI group (left fastigial nucleus lesion plus FNS plus MI). The serum expressions of acetylcholine (ACh), pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), and anti-inflammatory cytokines IL-10 were measured by ELISA. Subsequently, the infarct size, the infiltration of inflammatory cells, the fibrotic area, and cardiac function were also evaluated. Additionally, the expressions of the cholinergic anti-inflammatory pathway (CAP)-related proteins in infarct tissue, such as nuclear factor kappa B (NF-κB) and singal transducers and activators of transcription 3 (STAT3), were determined by Western blot. We found that FNS significantly increased ACh and IL-10 levels in serum, and decreased TNF-α and IL-6 levels. FNS significantly attenuated inflammatory cell infiltration, reduced infarct size, decreased fibrosis, increased left ventricular ejection fraction, and reduced mortality. Besides, the ratios of phosphorylated-STAT3/STAT3 and phosphorylated-NF-κB/NF-κB in infarct tissue significantly elevated after MI. FNS reduced the ratios of p-STAT3/STAT3 and p-NF-κB/NF-κB in infarct tissue. The protective effects of FNS were partially reversed by the fastigial nucleus lesion. Our data suggested that FNS can alleviate the inflammation after MI, and its cardiac neuroprotective mechanism may be achieved by increasing vagal tone, releasing ACh, and further activating the CAP via α7 nicotinic acetylcholine receptor. The precise mechanism remains to be elucidated.

The Smad3-dependent microRNA let-7i-5p promoted renal fibrosis in mice with unilateral ureteral obstruction.

Renal fibrosis is a common feature of all types of chronic kidney disease (CKD) and is tightly regulated by the TGF-β/Smad3 pathway. Let-7i-5p belongs to the let-7 microRNA family with diverse biological functions. It has been reported that let-7i-5p suppresses fibrotic disease in the heart, lungs, and blood vessels, while the role of let-7i-5p in renal fibrosis remains limited. In this study, we aimed to investigate the role of let-7i-5p in renal fibrosis in a mouse model of unilateral ureteral obstruction (UUO) and TGF-β1–stimulated renal tubular cell line TCMK1. The RNA-targeting CRISPR/Cas13d system was used to knock down let-7i-5p. Renal injury and fibrosis were determined by histological analysis, RT-PCR, Western blot, and immunostaining. Our results have shown that in the kidneys after UUO, the expression of let-7i-5p was significantly increased along with notable tubular injury and interstitial fibrosis. Electroporation of let-7i–targeting Cas13d plasmid efficiently knocked down let-7i-5p in kidneys after UUO with reduced tubular injury, fibrotic area, and expression of fibrotic marker genes α-SMA, fibronectin, and Col1a1. In TGF-β1–stimulated TCMK1 cells, knockdown of let-7i-5p by Cas13d plasmid transfection also blunted the expression of fibrotic marker genes. Most importantly, the genomic locus of let-7i showed enriched binding of Smad3 as revealed by chromatin immunoprecipitation. In TCMK1 cells, the overexpression of Smad3 can directly induce the expression of let-7i-5p. However, the deletion of Smad3 abolished TGF-β1–stimulated let-7i-5p expression. Collectively, these findings suggest that let-7i-5p is a Smad3-dependent microRNA that plays a pathogenic role in renal fibrosis. Let-7i-5p could be a promising target for the treatment of CKD-associated renal fibrosis.

Evaluation of the effects of Ankaferd haemostat application on bone regeneration in rats with calvarial defects: histochemical, immunohistochemical and scintigraphic study.

Bone wax, a haemostatic agent, is widely used in craniospinal surgical procedures for a long time, in spite of controversial results regarding its negative influence upon bone regeneration. In this experimental study, the effects of Ankaferd Blood Stopper (ABS), as an alternative haemostatic agent, were evaluated through histochemical, immunohistochemical and scintigraphic studies. The total of 30 adult female Wistar albino rats was randomly divided into three groups: intact control group (n = 10), bone wax group (n = 10), and ABS group (n = 10). Surgically, a 3.0 mm hole in diameter was drilled on the right side of calvarium of the rats using a Class Mini Grinder set in all three groups, as described previously. At the end of 8 weeks, bone healing and connective tissue alterations surrounding drilled calvarial defect areas of the rats were determined via haematoxylin and eosin and the Mallory's trichrome staining and anti-bone sialoprotein immunohistochemistry. Image Pro Express 4.5 programme was used for histomorphometric calculation of new bone and fibrotic tissue areas. All statistical analyses were made with SPSS 25.0 and analysis of variance (one-way ANOVA) followed by Bonferroni post hoc test was performed, p < 0.001 was considered as significance level. Histomorphometrically, it was found that he had the largest hole diameter and the least fibrotic scar area in the bone-wax group. In the bone wax group, it was observed that the material closed the hole and there was only a fibrotic scar tissue in the area between the bone tissue at the edge of the hole and bone wax, and a fibrotic tissue was formed in the bone wax area. During the histological procedure, this bone-wax material was poured and the sections were seen as a gap in this area. In the ABS haemostat group, the smallest hole diameter and the least fibrotic scar tissue were observed. Fibrotic scar tissue close to each other was found in the ABS haemostat and bone wax groups. Histological analysis of samples also showed a statistical significance for fibrotic connective tissue area between groups (p < 0.05). Scintigraphically, osteoblastic activity related to blood flow in the animal taken from the group with application of ABS haemostat was more pronounced compared to the other two groups. In our study, it has been concluded that the ABS yields affirmative effects on the bone healing, while bone wax leads to negative impact on the bone regeneration. Scintigraphic, histochemical and immunohistochemical data support the affirmative impact of the ABS haemostat application upon the bone regeneration apart from the quick stop of haemorrhage.

Circle Method for Robust Estimation of Local Conduction Velocity High-Density Maps From Optical Mapping Data: Characterization of Radiofrequency Ablation Sites.

Conduction velocity (CV) slowing is associated with atrial fibrillation (AF) and reentrant ventricular tachycardia (VT). Clinical electroanatomical mapping systems used to localize AF or VT sources as ablation targets remain limited by the number of measuring electrodes and signal processing methods to generate high-density local activation time (LAT) and CV maps of heterogeneous atrial or trabeculated ventricular endocardium. The morphology and amplitude of bipolar electrograms depend on the direction of propagating electrical wavefront, making identification of low-amplitude signal sources commonly associated with fibrotic area difficulty. In comparison, unipolar electrograms are not sensitive to wavefront direction, but measurements are susceptible to distal activity. This study proposes a method for local CV calculation from optical mapping measurements, termed the circle method (CM). The local CV is obtained as a weighted sum of CV values calculated along different chords spanning a circle of predefined radius centered at a CV measurement location. As a distinct maximum in LAT differences is along the chord normal to the propagating wavefront, the method is adaptive to the propagating wavefront direction changes, suitable for electrical conductivity characterization of heterogeneous myocardium. In numerical simulations, CM was validated characterizing modeled ablated areas as zones of distinct CV slowing. Experimentally, CM was used to characterize lesions created by radiofrequency ablation (RFA) on isolated hearts of rats, guinea pig, and explanted human hearts. To infer the depth of RFA-created lesions, excitation light bands of different penetration depths were used, and a beat-to-beat CV difference analysis was performed to identify CV alternans. Despite being limited to laboratory research, studies based on CM with optical mapping may lead to new translational insights into better-guided ablation therapies.

PSME4 Degrades Acetylated YAP1 in the Nucleus of Mesenchymal Stem Cells.

Intensive research has focused on minimizing the infarct area and stimulating endogenous regeneration after myocardial infarction. Our group previously elucidated that apicidin, a histone deacetylase (HDAC) inhibitor, robustly accelerates the cardiac commitment of naïve mesenchymal stem cells (MSCs) through acute loss of YAP1. Here, we propose the novel regulation of YAP1 in MSCs. We found that acute loss of YAP1 after apicidin treatment resulted in the mixed effects of transcriptional arrest and proteasomal degradation. Subcellular fractionation revealed that YAP1 was primarily localized in the cytoplasm. YAP1 was acutely relocalized into the nucleus and underwent proteasomal degradation. Interestingly, phosphor-S127 YAP1 was shuttled into the nucleus, suggesting that a mechanism other than phosphorylation governed the subcellular localization of YAP1. Apicidin successfully induced acetylation and subsequent dissociation of YAP1 from 14-3-3, an essential molecule for cytoplasmic restriction. HDAC6 regulated both acetylation and subcellular localization of YAP1. An acetylation-dead mutant of YAP1 retarded nuclear redistribution upon apicidin treatment. We failed to acquire convincing evidence for polyubiquitination-dependent degradation of YAP1, suggesting that a polyubiquitination-independent regulator determined YAP1 fate. Nuclear PSME4, a subunit of the 26 S proteasome, recognized and degraded acetyl YAP1 in the nucleus. MSCs from PSME4-null mice were injected into infarcted heart, and aberrant sudden death was observed. Injection of immortalized human MSCs after knocking down PSME4 failed to improve either cardiac function or the fibrotic scar area. Our data suggest that acetylation-dependent proteasome subunit PSME4 clears acetyl-YAP1 in response to apicidin treatment in the nucleus of MSCs.

Identifying Novel Genes and Variants in Immune and Coagulation Pathways Associated with Macular Degeneration

PurposeTo select individuals and families with a low genetic burden for age-related macular degeneration (AMD), to inform the clinical diagnosis of macular disorders, and to find novel genetic variants associated with maculopathies.DesignGenetic association study based on targeted and whole-exome sequencing.ParticipantsA total of 758 subjects (481 individuals with maculopathy and 277 controls), including 316 individuals in 72 families.MethodsWe focused on 150 genes involved in the complement, coagulation, and inflammatory pathways. Single-variant tests were performed on 7755 variants shared among ≥ 5 subjects using logistic regression. Gene-based tests were used to evaluate aggregate effects from rare and low-frequency variants (at minor allele frequency [MAF] ≤ 5% or ≤ 1%) in a gene using burden tests. For families whose affected members had a low burden of genetic risk based on known common and rare variants related to AMD, we searched for rare variants (MAF < 0.001) whose risk alleles occurred in ≥ 80% of affected individuals but not in controls. Immunohistochemistry was performed to determine the protein expression of a novel gene (coagulation factor II thrombin receptor-like 2 [F2RL2]) in retinal tissues.Main Outcome MeasuresGenotypes and phenotypes of macular degeneration.ResultsWe confirmed the association of a synonymous variant in complement factor H (Ala473, rs2274700, proxy to intronic rs1410996, r2 = 1) with maculopathy (odds ratio, 0.64; P = 4.5 × 10–4). Higher AMD polygenic risk scores (PRSs) were associated with intermediate and advanced AMD. Among families with low PRSs and no known rare variants for maculopathy, we identified 2 novel, highly penetrant missense rare variants in ADAM15, A disintegrin and metalloprotease, metallopeptidase domain 15 (p.Arg288Cys) and F2RL2 (p.Leu289Arg). Immunohistochemistry analyses revealed F2RL2 protein expression in cone photoreceptor outer segments and Müller glia cells of human and pig retinas. Coagulation factor II thrombin receptor-like 2 expression appeared increased in fibrotic areas in advanced AMD samples with neovascularization, suggesting that F2RL2 may play a role in the progression to advanced macular disease.ConclusionsNew missense rare variants in the genes ADAM15 and F2RL2 were associated with maculopathies. Results suggest that novel genes related to the coagulation and immune pathways may be involved in the pathogenesis of macular diseases.

Abstract P1135: Psme4 Degrades Acetylated Yap1 In The Nucleus Of Mesenchymal Stem Cells To Induce Cardiac Commitment

Intensive research has focused on minimizing the infarct area and stimulating endogenous regeneration after myocardial infarction. Our group elucidated that apicidin, a histone deacetylase (HDAC) inhibitor, robustly stimulates cardiac commitment of mesenchymal stem cells (MSCs) through acute loss of YAP1. Here we further studied the mechanism of this role of YAP1 in MSCs. We found that acute loss of YAP1 after apicidin treatment resulted in the mixed effects of transcriptional arrest and proteosomal degradation. Subcellular fractionation revealed that YAP1 was primarily localized in the cytoplasm. YAP1 was acutely relocalized into the nucleus and underwent proteosomal degradation. Interestingly, phosphor-S127 YAP1 was shuttled into the nucleus, suggesting that a mechanism other than phosphorylation governed subcellular localization of YAP1. Apicidin successfully induced acetylation and subsequent dissociation of YAP1 from 14-3-3, an essential molecule for cytoplasmic restriction. HDAC6 regulated both acetylation and subcellular localization of YAP1. An acetylation-dead mutant of YAP1 retarded nuclear redistribution upon apicidin treatment. We failed to acquire convincing evidence for polyubiquitination-dependent degradation of YAP1, suggesting that a polyubiquitination-independent regulator determined YAP1 fate. Nuclear PSME4, a subunit of the 26S proteasome, recognized and degraded acetyl YAP1 in the nucleus. MSCs from PSME4-null mice were injected into infarcted heart and aberrant sudden death was observed. Injection of immortalized human MSCs after knocking down PSME4 failed to improve either cardiac function or the fibrotic scar area. Our data suggest that acute ablation of YAP1 in the nucleus by the acetylation-dependent proteasome subunit PSME4 is mandatory for cardiac commitment of MSCs.

Abstract P1027: Cardioprotective Effects Of Genetically Engineered Cardiac Stem Cells By Spheroid Formation On Ischemic Cardiomyocytes

Background: Sca-1+ cardiac stem cells and their limited proliferative potential were major limiting factors for use in various studies. Methods: Therefore, the effects of sphere genetically engineered cardiac stem cells (S-GECS) inserted with telomerase reverse transcriptase (TERT) were investigated to examine cardiomyocyte survival under hypoxic conditions. GECS was obtained from hTERT-immortalized Sca-1+ cardiac stem cell (CSC) lines, and S-GECS were generated using poly-HEMA. Results: The optimal conditions for S-GECS was determined to be 1,052 GECS cells/mm 2 and a 48 hours culture period. Compared to adherent-GECS (A-GECS) and S-GECS showed significantly higher mRNA expression of the growth factors SDF-1α and CXCR4. S-GECS conditioned medium (CM) significantly reduced the proportion of early and late apoptotic cardiomyoblasts during CoCl 2 -induced hypoxic injury; however, gene silencing via CXCR4 siRNA deteriorated the protective effects of S-GECS against hypoxic injury. As downstream pathways of SDF-1α/CXCR4, the Erk and Akt signaling pathways were stimulated in the presence of S-GECS CM. S-GECS transplantation into a rat acute myocardial infarction model improved cardiac function and reduced the fibrotic area. These cardioprotective effects were confirmed to be related with the SDF-1α/CXCR4 pathway. Conclusions: Our findings suggest that paracrine factors secreted from transplanted cells may protect host cardiomyoblasts in the infarcted myocardium, contributing to beneficial left ventricle (LV) remodeling after acute myocardial infarction (AMI).

Abstract P2016: Gut Microbiota-derived Short-chain Fatty Acids Benefit Cardiac Extracellular Matrix Remodeling In Pressure Overload

Introduction: The gut microbiota has been implicated in various heart diseases by producing metabolites that modulate host immunity and metabolism. In our previous study, we found that mice with dysbiosis suffered more from cardiac rupture, resulting in higher mortality than mice possessing commensal gut flora after myocardial infarction. This suggests that there are unexplored routes to cardiovascular health through the gut microbiota. Hypothesis: We hypothesize that a community of symbiotic gut microbiota is required for heart mechanical modifications during adaptive cardiac remodeling under stress. Methods and Results: Pressure-overload stress was induced by a transverse aortic constriction (TAC) surgery and dysbiosis was induced by antibiotic treatment (ABX) in mice. Under echocardiography, ABX-TAC mice showed worse cardiac outcomes versus controls (ejection fraction = 59% ± 2% vs 67% ± 2%, P &lt; 0.001, N ≥ 12/group). Under microscopic examination of the extracellular matrix and tensile tests, ABX-TAC mice had larger fibrotic areas (9.4% ± 0.4% vs 5.7% ± 0.4%, P &lt; 0.0001) and collagen disarray, accompanied by more severe ventricular stiffening (Young’s moduli = 360 ± 10 kPa vs 280 ± 43 kPa, P &lt; 0.01, N ≥ 6/group). When establishing normal gut flora before surgery, germ-free mice had heart malfunctioning rescued (change of myocardial performance index = 15% ± 13% vs 120% ± 32% untreated, P &lt; 0.05, N ≥ 5/group). The normal gut flora was profiled by third-generation 16S sequencing to acquire bacterial information with high accuracy, followed by PICRUSt analysis revealing that the microbes favored the production of short-chain fatty acids (SCFAs) (N ≥ 8/group). When supplemented with SCFA before surgery, ABX mice gained better cardiac outcomes (ejection fraction = 65% ± 2% vs 53% ± 3% untreated, P &lt; 0.01, N ≥ 5/group). Cardiac fibroblasts treated with SCFA were less susceptible to TGF-β1-triggered fibrogenesis (COL1A1/GAPDH = 0.8 ± 0.3 vs 2.3 ± 0.3 untreated, P &lt; 0.01, n ≥ 4/group). Conclusions: In conclusion, our study demonstrates firstly that gut microbiota-derived SCFAs manipulate heart mechanical functioning under stress potentially acting via cardiac fibroblasts. This provides new insights into the management of heart diseases through the gut microbiota.

Use of wogonin as a cooperative drug with praziquantel to better combat schistosomiasis.

Schistosomiasis is one of the most devastating tropical diseases in the world. Currently, praziquantel (PZQ) represents the best pharmacological option for the treatment of schistosomiasis as it effectively kills the worm. However, the inability to reverse established liver damages often makes treatment futile. In the current study, we investigate whether combining the use of wogonin, a compound that was found to be liver-protective, with PZQ can attribute to the greatest beneficial effect in Schistosoma mansoni-infected mice. To determine the protective effect of PZQ-wogonin treatment on S. manosni-infected mice, histopathological analysis was done to evaluate the granuloma size and fibrotic areas in the liver. Western blotting was performed to analyze several injuries-related markers including fibrotic markers, inflammasomes, and apoptotic markers. Scanning electron microscopy was done to evaluate the effect of wogonin on the worms, and the worm and egg burden was calculated. Our results showed that PZQ-wogonin treatment significantly improved liver histopathology of S. mansoni-infected mice. Further analysis showed that PZQ-wogonin combinations are more effective in reducing fibrosis, inflammation, and apoptosis in the liver than that of individual drug use. Furthermore, our results revealed that wogonin is anthelmintic; and it works better with PZQ in reducing hepatic egg burden, further lessen the disease progression. In general, this combinatorial strategy may represent a new and effective approach to schistosomiasis treatment.