Transgenic Mice Research Articles

Establishment of a 3D organoid culture model for the investigation of adult slow-cycling putative intestinal stem cells.

The study of intestinal stem cells is a prerequisite for the development of therapies aimed at regenerating the gut. To enable investigation of adult slow-cycling H2B-GFP-retaining putative small intestinal (SI) stem cells in vitro, we have developed a three-dimensional (3D) SI organoid culture model based on the Tet-Op histone 2 B (H2B)-green fluorescent protein (GFP) fusion protein (Tet-Op-H2B-GFP) transgenic mouse. SI crypts were isolated from 6- to 12-week-old Tet-Op-H2B-GFP transgenic mice and cultured with appropriate growth factors and an animal-derived matrix (Matrigel). For in vitro transgene expression, doxycycline was added to the culture medium for 24h. By pulse-chase experiments, H2B-GFP expression and retention were assessed through direct GFP fluorescence observations, both by confocal and fluorescence microscopy and by immunohistochemistry. The percentages of H2B-GFP-retaining putative SI stem cells and H2B-GFP-retaining Paneth cells persisting in organoids were determined by scoring relevant GFP-positive cells. Our results indicate that 24 h exposure to doxycycline (pulse) induced ubiquitous expression of H2B-GFP in the SI organoids. During subsequent culture, in the absence of doxycycline (chase), there was a gradual loss (due to cell division) of H2B-GFP. At 6-day chase, slow-cycling H2B-GFP-retaining putative SI stem cells and H2B-GFP-retaining Paneth cells were detected in the SI organoids. The developed culture model allows detection of slow-cycling H2B-GFP-retaining putative SI stem cells and will enable the study of self-renewal and regeneration for further characterization of these cells.

Critical role of hepsin/TMPRSS1 in hearing and tectorial membrane morphogenesis: insights from transgenic mouse models

Mutations in various type II transmembrane serine protease (TMPRSS) family members are associated with non-syndromic hearing loss, with some mechanisms still unclear. For instance, the mechanism underlying profound hearing loss and tectorial membrane (TM) malformations in hepsin/TMPRSS1 knockout (KO) mice remains elusive. In this study, we confirmed significantly elevated hearing thresholds and abnormal TM morphology in hepsin KO mice, characterized by enlarged TM with gaps and detachment from the spiral limbus. Transgenic mouse lines were created to express either wild-type or a serine protease-dead mutant of human hepsin in the KO background. The Tg68;KO line, expressing moderate levels of wild-type human hepsin in the cochlea, showed partial restoration of hearing function. Conversely, the Tg5;KO or TgRS;KO lines, with undetectable hepsin or protease-dead hepsin, did not show such improvement. Histological analyses revealed that Tg68;KO mice, but not Tg5;KO or TgRS;KO mice, had a more compact TM structure, partially attached to the spiral limbus. These results indicate that hepsin expression levels correlate with improvements in hearing and TM morphology, and its protease activity is critical for these effects. Hepsin's role was further examined by studying its relationship with α-tectorin (TECTA) and β-tectorin (TECTB), non-collagenous proteins crucial for TM formation. Hepsin was co-expressed with TECTA and TECTB in the developing cochlear epithelium. Immunostaining showed decreased levels of TECTA and TECTB in hepsin KO TM, partially restored in Tg68;KO mice. These findings suggest that hepsin is essential for proper TM morphogenesis and auditory function, potentially by proteolytic processing/maturation of TECTA and TECTB and their incorporation into the TM.

BDNF and Cerebellar Ataxia.

Brain-derived neurotrophic factor (BDNF) has been proposed as a treatment for neurodegeneration, including diseases of the cerebellum, where BDNF levels or those of its main receptor, TrkB, are often diminished relative to controls, thereby serving as replacement therapy. Experimental evidence indicates that BDNF signaling countered cerebellar degeneration, sensorimotor deficits, or both, in transgenic ATXN1 mice mutated for ataxin-1, Cacna1a knock-in mice mutated for ataxin-6, mice injected with lentivectors encoding RNA sequences against human FXN into the cerebellar cortex, Kcnj6Wv (Weaver) mutant mice with granule cell degeneration, and rats with olivocerebellar transaction, similar to a BDNF-overexpressing transgenic line interbred with Cacng2stg mutant mice. In this regard, this study discusses whether BDNF is effective in cerebellar pathologies where BDNF levels are normal and whether it is effective in cases with combined cerebellar and basal ganglia damage.

Intranasal liposomal angiotensin-(1-7) administration reduces inflammation and viral load in the lungs during SARS-CoV-2 infection in K18-hACE2 transgenic mice.

To effectively reduce the health impact of coronavirus disease (COVID-19), it is essential to adopt comprehensive strategies to protect individuals from severe acute respiratory syndrome. In that sense, much effort has been devoted to the discovery and repurposing of effective antiviral and anti-inflammatory molecules. The endogenous peptide angiotensin-(1-7) [Ang-(1-7)] has been recently proposed as a promising anti-inflammatory agent to control respiratory infections. Liposomes also emerged as a safe and effective drug carrier system for local drug delivery to the lungs. In this context, the aim of this study was to develop a liposomal formulation of Ang-(1-7) [LAng (1-7)] and investigate its impact on animal survival as well as its antiviral and anti-inflammatory efficacies after intranasal administration in transgenic K18-hACE2 mice infected with SARS-CoV-2. The liposomal formulation was prepared by the ethanol injection method, exhibiting a mean diameter of 100 nm and a polydispersity index of 0.1. Following treatment of infected mice every 12 hours for 5 days, LAng (1-7) extended animal survival compared to the control groups that received either empty liposomes, free Ang-(1-7), or phosphate-buffered saline. Furthermore, the treatment with LAng (1-7) significantly decreased the viral load, as well as IL-6 and tumor necrosis factor levels in the lungs. Conventional treatment with remdesivir by parenteral route used as a positive control promoted similar effects, leading to improved survival rates and reduced viral load in the lungs without significant effects on IL-6 level. In conclusion, liposomal Ang-(1-7) emerges as a promising formulation to improve the treatment and decrease the severity of respiratory infections, such as COVID-19.

Small hepatitis B virus surface antigen (SHBs) induces dyslipidemia by suppressing apolipoprotein-AII expression through ER stress-mediated modulation of HNF4α and C/EBPγ.

Persistent infection with hepatitis B virus (HBV) often leads to disruptions in lipid metabolism. Apolipoprotein AII (apoAII) plays a crucial role in lipid metabolism and is implicated in various metabolic disorders. However, whether HBV could regulate apoAII and contribute to HBV-related dyslipidemia and the underlying mechanism remain unclear. This study revealed significant reductions in apoAII expression in HBV-expressing cell lines, the serum, and liver tissues of HBV-transgenic mice. The impact of HBV on apoAII is related to small hepatitis B virus surface antigen (SHBs). Overexpression of SHBs decreased apoAII levels in SHBs-expressing hepatoma cells, transgenic mice, and the serum of HBV-infected patients, whereas suppression of SHBs increased apoAII expression. Mechanistic investigations demonstrated that SHBs repressed the apoAII promoter activity through a HNF4α- and C/EBPγ-dependent manner; SHBs simultaneously upregulated C/EBPγ and downregulated HNF4α by inhibiting the PI3K/AKT signaling pathway through activating endoplasmic reticulum (ER) stress. Serum lipid profile assessments revealed notable decreases in high-density lipoprotein cholesterol (HDL-C), total cholesterol (TC), and triglycerides (TG) in SHBs-transgenic mice compared to control mice. However, concurrent overexpression of apoAII in these mice effectively counteracted these reductions in lipid levels. In HBV patients, SHBs levels were negatively correlated with serum levels of HDL-C, LDL-C, TC, and TG, whereas apoAII levels positively correlated with lipid content. This study underscores that SHBs contributes to dyslipidemia by suppressing the PI3K/AKT pathway via inducing ER stress, leading to altered expression of HNF4α and C/EBPγ, and subsequently reducing apoAII expression.IMPORTANCEThe significance of this study lies in its comprehensive examination of how the hepatitis B virus (HBV), specifically through its small hepatitis B virus surface antigen (SHBs), impacts lipid metabolism-a key aspect often disrupted by chronic HBV infection. By elucidating the role of SHBs in regulating apolipoprotein AII (apoAII), a critical player in lipid processes and associated metabolic disorders, this research provides insights into the molecular pathways contributing to HBV-related dyslipidemia. Discovering that SHBs downregulates apoAII through mechanisms involving the repression of the apoAII promoter via HNF4α and C/EBPγ, and the modulation of the PI3K/AKT signaling pathway via endoplasmic reticulum (ER) stress, adds critical knowledge to HBV pathogenesis. The research also shows an inverse correlation between SHBs expression and key lipid markers in HBV-infected individuals, suggesting that apoAII overexpression could counteract the lipid-altering effects of SHBs, offering new avenues for understanding and managing the metabolic implications of HBV infection.

Lipoprotein(a) induces spontaneous aortic dissection via increasing monocyte mediated vascular smooth muscle cell phenotype transformation in human Lp(a) transgenic mice

Abstract Background Elevated lipoprotein(a) [Lp(a)] is a prevalent, independent risk factor of atherosclerotic cardiovascular disease and aortic valve stenosis. Recent studies have reported that high levels of Lp(a) are strongly associated with aortic dissection (AD), independent of other cardiovascular risk factors. However, orthologues of Lp(a) are only found in humans, Old World monkeys, apes, and hedgehogs. The lack of a proper animal model has hindered elucidation of the true pathophysiological mechanisms of Lp(a). Here, we conducted the first humanized Lp(a) transgenic mice experiment to verify the pathological mechanism of Lp(a) in aortic dissection. Methods We conducted ApoE knock out and human ApoA transgenic mice via CRISPR-Cas9 and then crossed with human ApoB transgenic mice to achieve high levels of human-like Lp(a) particles in plasma. Plasma was assayed by ELISA for the concentrations of each lipoprotein. Echocardiography was performed to detect aortic morphology in vivo. Single cell transcriptome analysis was performed on the dissection lesion. Cell interaction was examined in macrophage from Lp(a) transgenic mice and vascular smooth muscle cell (VSMC) coculture system. Result Our results indicate that the concentration of ApoA and ApoB in plasma were increased in transgenic mice fed a chow diet. Low density lipoprotein (LDL) levels were increased and shows that LDL is the major cholesterol carrier in these mice. We were surprised to find that the dissection of the ascending aortic arch could be seen by echocardiography in transgenic mice as early as 2 months, and transgenic mice showed significant increase in aortic enlargement, dissection, and rupture in both the thoracic and abdominal aortic regions, resulting in a survival rate of only 58% after 12 months. Single cell transcriptome analysis revealed that Lp(a) transgene induced the inflammatory response, dedifferentiation and pathological VSMC phenotypical switch, and matrix metalloproteinase expression in macrophages. Monocytes isolated from transgenic mice with elevated Lp(a) remain in a long-lasting primed state, as evidenced by an increased capacity to transmigrate and produce proinflammatory cytokines on stimulation. Monocytes isolated from wild type mice cultured in media with high level Lp(a) for 24h also showed the ability to facilitated cocultured VSMC phenotype transformation, which was reversed by E06, a monoclonal antibody blocking the oxidized phospholipids in Lp(a). Conclusion These findings demonstrate that Lp(a) induces spontaneous aortic dissection via increasing monocyte trafficking to the arterial wall, mediating proinflammatory responses and VSMC phenotype transformation through its OxPL content. The study presents a rodent model of spontaneous aortic dissection and provide a novel mechanism by which Lp(a) mediates aortic dissection.Graphical abstract

Endothelial insulin-like growth factor-1 signaling regulates vascular barrier function and atherogenesis

Abstract Background Progressive deposition of cholesterol in the arterial wall characterizes atherosclerosis, which underpins most cases of myocardial infarction and stroke. Insulin-like growth factor-1 (IGF-1) is a hormone that regulates systemic growth and metabolism and possesses anti-atherosclerotic properties. However, the role of IGF-1 in the endothelium remains unexplored. Purpose To study the effect of increased endothelial IGF-1 receptor (IGF-1R) expression on atherogenesis using in vivo and in vitro models. Methods We generated atherosclerosis prone ApoE-/- transgenic mice with endothelium restricted overexpression of human IGF-1R (hIGFREO-ApoE-/-) or signalling defective K1003R mutant IGF-1R (mIGFREO-ApoE-/-). ApoE-/- littermates were controls. Following 12 weeks of western diet, we assessed atherosclerosis, systemic metabolism, leukocyte homeostasis and vascular permeability using histology, flow cytometry, metabolic testing, bone marrow transplantation and in vivo perfusion with either VE-Cadherin, Evans blue or BODIPY-LDL. For in vitro studies, human umbilical vein endothelial cells were transduced with custom-made lentivirus particles allowing doxycyline-inducible wild-type or mutant K1003R IGF-1R overexpression; with transduced cells not exposed to doxycycline as controls. Confluent cells were exposed to 100mM hydrogen peroxide to model the permeability-inducing conditions observed in atherosclerosis. In vitro, we assessed junctional proteins, FITC-dextran permeability and transcellular BODIPY-LDL uptake. Results We show that mice with endothelial overexpression of IGF-1R have less atherosclerosis, arterial cholesterol uptake, and vascular leakage of multiple organs. Conversely, mice with endothelial overexpression of signalling defective IGF-1R did not exhibit these phenomena. We found no differences in systemic metabolism or growth. In complementary in vitro studies, overexpressing wildtype IGF-1R altered the localization of some tight junction proteins (VE-Cadherin and Claudin 5) and reduced leakage across human endothelial monolayers; this was not observed with signalling defective IGF-1R. Moreover, it reduced endothelial cell internalization of cholesterol-rich lipoproteins and increased the association of these particles with clathrin, but not caveolin-1, both of which participate in vesicular uptake of lipoproteins. Conclusion Overall, our findings indicate that endothelial IGF-1 signalling modulates both para- and trans-cellular vascular barrier function. This is particularly relevant to the phenomenon of atherosclerosis and suggests that increased vascular IGF-1 signalling reduces atherosclerosis. Beyond this, our data are potentially relevant to many disease processes associated with altered vascular barrier function. This discovery could lead to novel therapeutics to normalise vascular barrier function.

Huang-Pu-Tong-Qiao Formula Alleviates Hippocampal Neuron Damage by Inhibiting NLRP3 Inflammasome-mediated Pyroptosis in Alzheimer's Disease.

Huang-Pu-Tong-Qiao (HPTQ), a Traditional Chinese Medicine formula, has achieved remarkable efficacy in clinically treating Alzheimer's disease (AD). Pyroptosis refers to the inflammatory necrosis of cells, which contributes to AD pathological progression. However, it is unclear whether the therapeutic effect of HPTQ on AD is related to reducing pyroptosis. In this study, the network pharmacology analysis was used to predict the molecular mechanism of HPTQ in treating AD and validated our hypothesis through mice and cell experiments. APP/PS1 transgenic mice and Aβ25-35-injured HT22 cells were used as AD models in vivo and in vitro. The pharmacological effects and mechanisms of HPTQ on AD were evaluated by Morris water maze, Y-maze, transmission electron microscope, immunofluorescence, Hoechst/PI staining, western blot, and ELISA. Network pharmacology reveals the correlation between the therapeutic effect of HPTQ on AD and the NOD-like receptor signaling pathway. In APP/PS1 mice, HPTQ reduced the escape latency and maintained cell membrane integrity. In HT22 cells, 15% HPTQ-medicated serum and 10 µM MCC950 increased cell viability and decreased PI positive rate compared with the Model group. In addition, HPTQ treatment in AD animal and cell models reduced the protein expressions of NLRP3, ASC, cleaved caspase-1, GSDMD, GSDMD-N, IL-1β, and IL-18. The experimental results of MCC950 specifically inhibiting the NLRP3 expression suggested that HPTQ might reduce neuronal pyroptosis by reducing NLRP3 inflammasome. Network pharmacology and experimental validation suggested that HPTQ alleviated NLRP3 inflammasome-mediated neuronal pyroptosis in AD, which could provide valuable candidate drugs for AD clinical treatment.

Role of inflammation and macrophages in the formation of the atrial cardiomyopathy associated with obesity

Abstract Introduction The atrial fibrillation (AF), the most frequent cardiac arrhythmia in clinical practice is often associated with an atrial cardiomyopathy (ACM) which is favoured by several clinical conditions including metabolic diseases such as obesity. Recently, the epicardial adipose tissue (EAT) has emerged as an important component of ACM notably in the context of metabolic diseases. Epicardial progenitor cells (EPC) are the main source of EAT. The replacement of EAT by fibrosis is arrhythmogenic and involved inflammation but not yet characterized. The hypothesis tested here is that macrophages are central players for both the recruitment of EPC and replacement of EAT by fibrosis. Purpose-Aims First to characterize subpopulation of macrophages involves in the progression of ACM. Second, to study how the inflammatory environment regulates EPC differentiation. Method A spatial gene expression was used to analyze human samples (n=4) (Agreement, CODECOH, Declaration or authorization of activities involving the preservation and preparation of human body parts for scientific purposes DC-2020-4183). A well-characterized mouse model of atrial cardiomyopathy associated with obesity induced by high fat diet (HFD) was used to characterize macrophages associated with progression of ACM by using immunolabeling together with single nuclei RNA sequencing approaches. ACM was characterized by using echocardiography and atrial burst pacing to evaluate determine AF vulnerability. Transgenic RGB-MAC mice with macrophages tagged with McApple-Csf1R and ECGFP-CX3CR1 or with a knock-out for CCR2 were used to follow infiltration of myeloid cells in the atrial at the different stages of the ACM. Bi-photon microscopy was used to visualize cell subpopulations in the distinct area of the atrial wall. In some experiments EPC and macrophages were cocultured with transwell. Results Macrophages were detected in subepicardium of human atrial samples in area of fibro-fatty infiltrates. After 2 months of HFD, monocytes were infiltrated atria and macrophages were overexpressed in atria from 1 to 4 months of HFD. Using Bi-photon microscopy it was possible to visualise resident ECGFP-CX3CR1+ macrophages at the contact of EAT whereas McApple-Csfr1r+ macrophages were at the junction with fibrosis (figure 1). Preliminary data generated by single nuclei RNA sequencing showed clusters of different subpopulations of macrophages at different time point of the ACM (figure 2). We will present results on the effects of suppression of the CCR2 subpopulation of macrophages on characteristics of the ACM. Conclusion Distinct populations of macrophages i.e resident and monocyte-derived macrophages, participate to the distinct stages of the ACM caused by obesity in mice.Myeloid cells infiltration in epicardiumCCR2+cells infiltration in EAT-fibrosis

NCOR1 orchestrates macrophage inflammation in heart failure with preserved ejection fraction: a translational study

Abstract Background/Introduction Inflammatory response is a key player in heart failure with preserved ejection fraction (HFpEF) but the underlying mechanisms are poorly understood. Recent studies have shown a critical involvement of cardiac macrophage activation in myocardial remodeling thus contributing to HFpEF. Purpose To investigate the role of cardiac macrophage inflammation in experimental and human HFpEF. Methods We employed a translational approach combining human and murine LV myocardial tissue, FACS analysis of cardiac immune cells, in vitro experiments in RAW 264.7 cells exposed to metabolic stress by palmitic acid (PA), bone marrow-derived macrophages from healthy and HFpEF mice, cardiomyocytes (H9c2), endothelial cells (HAECs) as well as ex vivo functional analyses in LV tissues. Human LV myocardial samples were used to evaluate the pro-inflammatory activity of macrophages in the HFpEF myocardium. Induction of HFpEF in mice was done through a validated mouse model combining metabolic (high-fat diet) and hemodynamic stress (L-NAME). Cardiac function was investigated by high resolution ultrasound imaging (Vevo3100) and the LV myocardial samples were used to study the macrophage polarization and their inflammatory activity. Based on interrogation of available RNA-seq datasets – showing the transcriptional repressor NCOR1 as a relevant signature in macrophages during cardiac inflammation, we generated myeloid cell-specific NCOR1 knockout (Ncor1-KO) transgenic mice to investigate the in vivo effects of NCOR1 deletion on the HFpEF phenotype. Results The expression of macrophage markers in human LV specimens showed an increase in pro-inflammatory macrophages (M1) and a decrease in regulatory macrophages (M2) as compared to age-matched control mice. HFpEF mice showed increased expression of vascular adhesion molecules (ICAM1, ICAM2, E-selectin) and a significant recruitment of M1 macrophages as proved by qPCR, WB, and flow cytometry analysis. Of interest, diastolic dysfunction - assessed by E/A ratio and isovolumic relaxation time (IVRT) – and lung congestion were significantly reduced in mice with macrophage NCOR1 deletion as compared to WT littermates, while exercise tolerance was significantly improved. These findings were associated with a reduction of myocardial inflammation (TNF-a, IL6, IL-1b) in Ncor1-KO mice. In vitro assays showed that stimulation of macrophages with PA induced M1 type switch and increased NCOR1, TNF-a, IL-6, and IL-1b levels, whereas NCOR1 depletion prevented detrimental changes of macrophage secretome (Fig.2). Conclusion NCOR1 is a crucial regulator of immune response and macrophage inflammation in the heart. Our findings highlight its potential as a target for therapies aimed at preventing myocardial damage in HFpEF.

SRAGE attenuates myocardial ischemia-reperfusion injury by recruiting Tregs through chemokine CCL4

Abstract Background Immune response has recently been shown to play a crucial role in myocardial ischemia-reperfusion (MIR). Our previous studies have confirmed that soluble receptors for advanced glycation end-products (sRAGE) could reduce myocardial ischemia-reperfusion injury (MIRI) by increasing the number of regulatory T cells (Tregs). However, the precise mechanism is still incompletely understood. Purpose The present study aimed to determine which kind of chemokines play a major role in recruiting Tregs during MIRI. The potential mechanism of sRAGE on the chemokine production was further explored. Methods We modeled mice with cardiac-specific overexpression of sRAGE by crossing the floxed sRAGE mouse model with the cardiomyocyte-specific Cre transgenic mouse. Experimental models were established utilizing left anterior descending coronary artery ligation in mice and oxygen-glucose deprivation/reoxygenation (OGD/R) in vitro. Transcriptomics, quantitative real-time polymerase chain reaction, immunohistochemistry, flow cytometry, and western blotting were used to determine the chemokines and related pathways involved in the recruitment of Tregs during MIRI. Results Using transcriptomics, quantitative real-time polymerase chain reaction, and western blotting approach, we identify the chemokine CCL4 plays a crucial role during regulatory T cell recruitment in the heart after MIRI. In vivo studies show that depletion of CCL4 reduces Tregs infiltration into the ischemic myocardium, subsequently deteriorating cardiac injury post-MIR. Mechanistically, sRAGE promotes cardiomyocytes to express and secrete CCL4 via the JNK/AP-1 pathway in vitro. Selective inhibition of the JNK/AP-1 pathway could decrease CCL4 production in sRAGE overexpression cardiomyocytes. Conclusions sRAGE induces Tregs recruitment and alleviates MIRI via the JNK/AP-1/CCL4 pathway, preserving myocardial function and impeding inflammation and apoptosis, providing new perspectives for immunotherapeutic targets in MIRI treatment.

STIL overexpression shortens lifespan and reduces tumor formation in mice.

Centrosomes are the major microtubule organizing centers of animal cells. Supernumerary centrosomes are a common feature of human tumors and associated with karyotype abnormalities and aggressive disease, but whether they are cause or consequence of cancer remains controversial. Here, we analyzed the consequences of centrosome amplification by generating transgenic mice in which centrosome numbers can be increased by overexpression of the structural centrosome protein STIL. We show that STIL overexpression induces centrosome amplification and aneuploidy, leading to senescence, apoptosis, and impaired proliferation in mouse embryonic fibroblasts, and microcephaly with increased perinatal lethality and shortened lifespan in mice. Importantly, both overall tumor formation in mice with constitutive, global STIL overexpression and chemical skin carcinogenesis in animals with inducible, skin-specific STIL overexpression were reduced, an effect that was not rescued by concomitant interference with p53 function. These results suggest that supernumerary centrosomes impair proliferation in vitro as well as in vivo, resulting in reduced lifespan and delayed spontaneous as well as carcinogen-induced tumor formation.

Cardioprotective effects of nicorandil on mitochondrial apoptosis signaling in the failing heart of cardiac-specific Bcl-2-associated athanogene (BAG) 3 knockout mice

Abstract Background Bcl-2-associated athanogene (BAG) 3 is a known regulator of mitochondrial apoptotic cell death and autophagic protein degradation in the heart. Disruption of the BAG3 gene in a cardiac-specific manner can lead to cardiac failure in mice. However, therapeutic approaches for cardiac failure induced by BAG3 gene disruption remain poorly understood. Method and results We characterized mice with the ablated BAG3 gene using the Cre-recombinase-loxp site system in a cardiac-specific manner (BAG3f/f crossbred with cardiac specific Cre transgenic mice; BAG3 cKO) and examined the therapeutic effect of nicorandil, an antianginal drug, in BAG3 cKO mice. Cardiac BAG3 levels were markedly reduced in the cytoplasmic and mitochondrial fractions from the hearts of BAG3 cKO mice compared to BAG3f/f mice. BAG3cKO mice exhibited cardiac disease such as a reduction in fractional shortening detected by echocardiography, cardiac fibrosis at 6 months of age, and approximately 40% premature death by 6 months of age. Electron microscopy analysis confirmed cardiomyocyte loss and mitochondrial abnormalities in some areas of the heart section from BAG3cKO mice at 4.5 months of age. A marked decrease in the level of cytochrome C in the mitochondrial fraction and a slight increase in the cytoplasmic fraction, along with apoptotic cell death, were observed in hearts from BAG3 cKO mice at 4.5 months. Mitochondrial levels of anti-apoptotic factors, such as Bcl-2 and Bcl-xl, were reduced, while there was a marked increase in Bak1, an apoptotic inducible factor, in hearts from BAG3 cKO mice at 4.5 months of age. These results suggest that cardiac mitochondrial impairment and subsequent apoptotic cell death occurred in BAG3cKO mice. Nicorandil treatment (81 mg/L drinking water) initiated at 2 months of age prevented the reduction of fractional shortening, cardiac fibrosis, ultrastructural cardiac abnormalities, and premature death. Furthermore, nicorandil treatment prevented the decrease in mitochondrial cytochrome C and the increase in cytoplasmic cytochrome C in hearts from BAG3 cKO mice at 4.5 months of age. Nicorandil treatment also inhibited the elevation of mitochondrial Bak1 levels, reduction in Bcl-2 levels, and occurrence of apoptotic cell death in hearts from BAG3 cKO mice at 4.5 months of age. Conclusion Long-term treatment with nicorandil can inhibit cardiac disease induced by cardiac- specific BAG3 ablation through mitochondrial protection via the prevention of activation of mitochondrial apoptosis signaling.

Epi-Allele: CRISPR-mediated allele-specific epigenetic editing alleviates hypertrophic cardiomyopathy

Abstract Background Hypertrophic cardiomyopathy (HCM) is a prevalent genetic heart disease caused by variants in sarcomere genes like MYH7, which accounts for 40% of cases with 75% being heterozygous. HCM shows incomplete penetrance, where mutant allele expression correlates with severity. Thus, allele-specific suppression is a potential therapy. While allele-specific RNAi or CRISPR/Cas9 showed moderate efficacy, low expression level of MYH7 is associated with the risk of heart failure and dilated cardiomyopathy. We hypothesized CRISPR-based epigenetic editing（dCas9 fused with DNA methylation and histone regulation elements） may potently achieve phenotypic modification without impacting overall gene expression. Objective We investigated allele-specific CRISPR epigenetic editing (Epi-Allele) to selectively suppress mutant Myh6 allele expression without affecting total expression, as a novel HCM therapy in a mouse model. Results We first generated C57R403Q/DBA mice to simulate heterozygous HCM patients. Allele-specific Myh6 gRNAs reduced C57 allele expression and increased DBA expression without impacting total Myh6 expression in primary cardiomyocytes, effects sustained for one year in report cells. HCM mice with Epigenetic editor and allele-specific gRNAs (Epi-Allele) showed reduced left ventricular wall thickness, normalized ECG, and less cardiac fibrosis. Similar therapeutic effects were observed in HCM mice treated by a dual AAV delivery system of Epi-Allele. We further found that Epi-Allele could reverse established disease in the inducible transgene mice. We’ve also seen similar surprising therapeutic effects from Epi-Alleles in patient-derived pluripotent stem cell-induced cardiomyocytes (iPSC-CMs). Conclusion We suggest that Epi-Allele may be a novel therapy for HCM and other dominant diseases where the mutant gene plays a vital role, overcoming the limitations of previous allele-specific suppression strategy.Figure abstract of Epi-AlleleThe universality of Epi-Alleles

Lipin1 prevents ischemic heart disease by regulating lipid homeostasis

Abstract Background Lipid metabolism plays a crucial role in the pathophysiology of cardiovascular diseases. Lipin1 plays a pivotal role in lipid metabolism regulation. In the nucleus, Lipin1 acts as a transcriptional co-stimulatory molecule, interacting with peroxisome proliferator–activated receptor–gamma coactivator 1 alpha (PGC1α) and peroxisome proliferator–activated receptor alpha (PPARα). In the cytoplasm, Lipin1 functions as a phosphatidate phosphatase, converting phosphatidic acid to diacylglycerol to regulate lipid metabolism. Low expression of Lipin1 has been implicated in the severity of cardiac dysfunction in mice. Purpose This study aimed to investigate the role of Lipin1 in the cardiac remodeling after myocardial infarction (MI), revealing its impact on lipid metabolism and molecular pathway associated with cardiac dysfunction. Methods Two types of transgenic mice, cardiomyocyte-specific Lipin1 knockout (cKO) and overexpression (cOE) mice were used in this study. Eight to ten-week-old male mice were subjected to MI by coronary artery ligation. Cardiac lipid droplets were quantified, and triacylglycerol and free fatty acid levels were measured one week after MI surgery. Four weeks after MI, cardiac morphology and function were evaluated using echocardiography, alongside measurements of body weight (BW) and heart weight (HW). Additionally, gene expression analysis and histological evaluation were conducted to assess fatty acid metabolism, cardiac fibrosis, inflammation, and oxidative damage. Results Before MI surgery, there were no differences in HW/BW ratio, left ventricular end-diastolic diameter (LVDd) or LV fractional area change (LVFAC) among cKO/ cOE mice and their littermate controls. After MI, cKO mice exhibited significant cardiac dysfunction with larger LVDd, lower LVFAC and increased HW/BW ratio compared to littermate controls. This was accompanied by exacerbated cardiac fibrosis, increased inflammation, and augmented reactive oxygen species accumulation compared with their controls. Conversely, cOE mice displayed improved LVFAC, reduced cardiac fibrosis, inflammation, and reactive oxygen species accumulation compared to their littermate controls. Notably, the transgene of Lipin1 induced changes in the number of cardiac lipid droplets (LDs). cKO mice hearts showed a reduction in cardiac LDs with decreased levels of triacylglycerol and free fatty acids compared to the littermate controls. In contrast, cOE mice displayed increased cardiac LDs and upregulated expression of fatty acid metabolism–related genes after MI. Conclusion These results suggested that Lipin1 has a protective role against ischemic injury through controlling lipid metabolism in ischemic cardiomyocytes. Thus, Lipin1 emerges as a potential therapeutic target for myocardial infarction.

An epigenetic editor targeting human PCSK9 efficiently and durably lowers Low Density Lipoprotein Cholesterol (LDL-C) in non-human primates

Abstract Background Reducing the risk of atherosclerotic cardiovascular disease is dependent on both the magnitude and cumulative duration of LDL-C lowering. However, in clinical practice achieving treatment goals is often hampered by low adherence to standard of care. Although PCSK9 inhibitors represent an effective option for LDL-C lowering, they require chronic life-long treatment. Epigenetic editing is a new therapeutic approach designed to durably silence genes by leveraging nature’s endogenous cellular mechanism for gene regulation via CpG methylation without the inherent genotoxic risks of genome editing approaches that nick or cut the DNA. We are developing a PCSK9 epigenetic editor (PCSK9-EE) to durably silence PCSK9, with the promise of lifelong reduction in LDL-C. Methods Here, we describe the development of an epigenetic editor targeting human PCSK9. This epigenetic editor consists of a DNA targeting component fused to a transcriptional repressor domain and a DNA methyltransferase domain. PCSK9-EE was first screened and evaluated for specificity and efficacy in primary human hepatocytes (PHH). In vivo efficacy and durability were then evaluated in a transgenic mouse carrying the human PCSK9 genomic locus (hPCSK9-Tg mouse) and non-human primates (NHP). Results Our initial screen of PCSK9-EEs identified several hits that efficiently suppressed secreted PCSK9 levels in immortalized liver cells. We confirmed that our top PCSK9-EE candidates robustly decreased PCSK9 secretion in PHH and were found to be highly specific at targeting PCSK9 as assessed by RNA-seq, methylation array, and whole-genome methylation sequencing. To examine in vivo activity, we treated hPCSK9-Tg mice with a single administration of a lipid nanoparticle delivering mRNA encoding our PCSK9-EE and observed >97% reduction in liver PCSK9 mRNA, and >98% reduction in plasma PCSK9 for the duration of the study (one year). We then delivered PCSK9-EE in NHP and observed a robust reduction in plasma PCSK9 with a concomitant reduction in LDL-C. These effects were found to be durable for at least 6 months following a single administration of PCSK9-EE in NHP. To establish a mechanistic relationship between PCSK9-EE’s molecular action and durable PCSK9 silencing, we performed serial liver biopsies in each NHP approximately 2 months apart. Robust CpG methylation at the PCSK9 genomic locus was observed in the first liver biopsy and was comparable to that observed in the second liver biopsy suggesting that PCSK9-EE induced stable CpG methylation in NHP livers. Conclusions We believe PCSK9-EE may offer an effective, safe, and durable approach to suppress hepatic PCSK9 expression and achieve long-lasting reduction in LDL-C. Furthermore, epigenetic editing may hold promise for one-and-done approaches for the treatment of atherosclerotic cardiovascular disease without cutting, nicking, or altering the DNA sequence.

Combination therapy with GalNAc-siRNA drugs targeting both PCSK9 and APOC3 resulted in enhanced lipid lowering in mice

Abstract Introduction Aggressive lowering of both atherogenic low-density lipoprotein cholesterol (LDL-C) and triglyceride-rich lipoproteins (TRLs) could reduce residual atherosclerotic cardiovascular disease (ASCVD) risk. Drugs targeting PCSK9 or APOC3 have shown good lipid-lowering effects in clinic. Here, we have developed GalNAc-conjugated siRNAs, RBD7022 targeting PCSK9 and RBD5044 targeting APOC3, as novel therapeutic agents to lower LDL-C and triglyceride (TG) plasma levels respectively. Each drug shows potent and durable effect in silencing its own target. However, it is unknown whether the combined therapy with siRNA drugs targeting both PCSK9 and APOC3 will generate enhanced effects in lipid lowering. Purpose To investigate the effects of fixed-dose combination of RBD7022 and RBD5044 on LDL-C, TG and total cholesterol (TC). Methods RBD7022 and RBD5044 completely match the human PCSK9 and APOC3 transcripts respectively. RBD7022 has 2 nucleotide mismatches with mouse pcsk9, but still shows silencing activity in mice, while RBD5044 has no activity due to 5 mismatches with mouse apoc3. Therefore, humanized APOC3 transgenic mice were used in this study, which had serious hypertriglyceridemia with relatively high cholesterol and TG levels. They were randomized into 8 groups with treatment of RBD7022 (1 mg/kg or 3 mg/kg), RBD5044 (3 mg/kg or 6 mg/kg), or 3 different dose combinations of RBD7022 and RBD5044, as well as PBS control. All animals were administrated once on Day 1. Plasma was collected to quantify lipid biomarkers. Results RBD7022 showed a robust effect on LDL-C reduction, with inhibition rates of 45% in 3 mg/kg and 43% in 6 mg/kg, indicating 3 mg/kg was a saturated dose for RBD7022 in LDL-C lowering. RBD5044 showed a dose-dependent effect on LDL-C reduction, with inhibition rate of 52% at 1 mg/kg and 59% at 3 mg/kg. Interestingly, significantly enhanced LDL-C reduction was observed in the combination groups, with 67% reduction in LDL-C at RBD5044 1 mg/kg plus RBD7022 3 mg/kg, 73% reduction in LDL-C at RBD5044 3 mg/kg plus RBD7022 3 mg/kg and 78% reduction in LDL-C at RBD5044 3 mg/kg plus RBD7022 6 mg/kg, showing dose-dependent effects (Figure 1A). Enhanced TC reduction was also found in combination at RBD5044 3 mg/kg plus RBD7022 6 mg/kg compared with RBD5044 or RBD7022 alone. RBD5044 showed a dose dependent effect on TG reduction with inhibition rates of 56% at 1 mg/kg and 83% at 3 mg/kg. RBD7022 treatment alone did not show significant effect on TG reduction. No added effects of RBD7022 on TG levels was found in the combination with RBD5044, indicating the TG lowering was induced by RBD5044. Conclusions The combination of RBD7022 targeting PCSK9 and RBD5044 targeting APOC3 simultaneously reduce LDL-C and TC levels. The combination of RBD7022 and RBD5044 could be a better therapeutic option to further reduce LDL-C, particularly for mixed hyperlipidemia patients and other high-risk individuals with both high LDL-C and high TG levels.Figure 1

Abnormal outer and inner retina in a mouse model of Huntington’s disease with age

Huntington’s disease (HD) is a progressive neurodegenerative disorder characterized by motor dysfunction and cognitive decline. While retinal abnormalities have been documented in some HD patients and animal models, the nature of these abnormalities—specifically whether they originate in the inner or outer retina—remains unclear, particularly regarding their progression with age. This study investigates the retinal structure and function in HD transgenic mice (R6/1) compared to C57BL/6 J control mice at 2, 4, and 6 months of age, encompassing both pre-symptomatic and symptomatic stages of HD. Pathological assessments of the striatum and evaluations of motor function confirmed significant HD-related alterations in R6/1 mice at 6 months. Visual function was subsequently analyzed, accompanied by immunofluorescent staining of retinal and optic nerve tissues over time. Our findings revealed that R6/1 mice exhibited pronounced HD symptoms at 6 months, characterized by neuronal loss in the striatum and impaired locomotor abilities. Functionally, visual acuity declined at 6 months, while retinal light responses began to deteriorate by 4 months. Structurally, R6/1 mice demonstrated a global reduction in cone opsin expression as early as 2 months, with a decrease in rhodopsin levels at 4 months, alongside a thinner retinal structure compared to controls. Notably, rod bipolar cell populations were decreased at 6 months, exhibiting shorter dendritic branches and reduced synaptic connections with photoreceptors in the outer retina. Additionally, ganglion cell numbers in the inner retina decreased at 6 months, accompanied by aberrant neural fibers in the optic nerve. Microglial activation was evident at 4 months, while astrocytic activation was observed at 6 months. Aggregates of mutant huntingtin (mHTT) were first detected in the ganglion cell layer and optic nerve at 2 months, subsequently disseminating throughout all retinal layers with advancing age. These results indicate that retinal pathology in R6/1 mice manifests earlier in the outer retina than in the inner retina, which does not align with the progression of mHTT aggregation. Consequently, the R6/1 mouse retina may serve as a more effective model for elucidating the mechanisms underlying HD and evaluating potential therapeutic strategies, rather than functioning as an early diagnostic tool for the disease.

Fibroblast-specific deletion of ERBB4 impairs cardiac regeneration in neonatal mice

Abstract Background Scarless regeneration of the neonatal mammalian heart is a remarkable but poorly understood process. Previous studies have shown that paracrine actions of neuregulin-1 (NRG1) are indispensable to stimulate cardiomyocyte proliferation in the injured neonatal heart, and are mediated by activation of the ERBB4 receptor. It remains unclear, however, whether NRG1 also contributes to other steps of neonatal cardiac regeneration, e.g. to the control of collagen synthesis by fibroblasts, hence contributing to scarless healing. Purpose To study temporal evolution of the infarct size during 21 days after myocardial infarction (MI) in neonatal fibroblast-specific Erbb4 knock-out (KO) and wild type (WT) mice. We hypothesized that scarless healing would be completed within 21 days in WT mice, and incomplete in KO mice. Methods Fibroblast-specific hemizygous Erbb4 KO (FB-Erbb4-KO) mice were generated (Erbb4F/+ Col1a2-Cre+, C57BL/6 background), because fibroblast-specific nullizygous Erbb4 KO were not viable. Validation of the KO was performed both on RNA (qPCR) and protein (western blot) level. MI was induced in one-day old FB-Erbb4-KO and WT littermates of both sexes by permanent ligation of the left anterior descending (LAD) coronary artery under hypothermic anaesthesia. Pups from both groups were euthanized at 4 (WT, n=9; KO, n=11), 7 (WT, n=12; KO, n=4), 10 (WT, n=11; KO, n=7) and 21 (WT, n=16; KO, n=8) days post-MI. Following dissection, hearts were formalin-fixed and paraffin embedded for histopathological evaluation. Transverse sections were cut at 4 distinct locations from the ligation site to the apex and stained with Masson’s Trichrome for infarct scar measurement. Genotyping was performed post-mortem and unblinded after data analysis. Results Both the transgenic mice model and the LAD ligation technique were successfully validated. No significant differences in the initial infarct scar size were observed between WT and FB-Erbb4-KO mice 4 days post-MI (p=0.6556). 7-days post-MI the infarct scar size was 6-fold smaller compared to 4 days post-MI in both FB-Erbb4-KO and WT mice, with a trend towards a larger infarct scar size in the FB-Erbb4-KO group compared to WT (p=0.0989). At day 10 post-MI, infarct scar size in the FB-Erbb4-KO group was significantly larger (p=0.0499) compared to WT. At 21 days post-MI, the infarct size had almost completely disappeared in the WT group, but remained clearly present in the FB-Erbb4-KO group (Fig. 1, p=0.0099). Infarct scar sizes at all timepoints are graphically shown in Fig. 2. Conclusion Fibroblast-specific deletion of the ERBB4 receptor causes incomplete resolution of the infarct scar in murine neonates 21 days after MI. While the initial infarct scar size 4 days after injury is the same, impaired scar resolution becomes progressively more evident towards day 21 post-MI. These findings indicate that scarless healing of the neonatal mammalian heart requires NRG1/ERBB4 signalling in fibroblasts.Fig. 1Fig. 2

Bay 11-7082, an NF-κB Inhibitor, Prevents Post-Inflammatory Hyperpigmentation Through Inhibition of Inflammation and Melanogenesis.

Post-inflammatory hyperpigmentation (PIH) is a very common disorder of cutaneous hyperpigmentation, which poses a persistent management challenge in the fields of dermatology and esthetics. This study was designed to explore the anti-melanogenic and anti-inflammatory effects of Bay 11-7082, an NF-κB inhibitor, using small-molecule screening, to determine its potential application for PIH prevention. The molecular mechanisms were investigated invitro and exvivo in epidermis-humanized mice using melanin content, RT-PCR, and immunoblotting. Bay 11-7082 suppressed proinflammatory cytokines and ameliorated 2,4-dinitrofluorobenzene (DNFB)-induced contact dermatitis on day 15. The suppression of melanin synthesis by Bay 11-7082 was attributed to the reduction of MITF, which was induced by extracellular signal-regulated kinase activation. Bay 11-7082 reduced epidermal melanin accumulation in UVB-stimulated exvivo human epidermis as well as in the ear and tail skin of K14-stem cell factor (SCF) transgenic mice. Topical administration of Bay 11-7082 improved PIH on day 35 in the post-DNFB dorsal skin of K14-SCF transgenic mice. In conclusion, Bay 11-7082 can be considered a promising candidate for the development of a preventive topical agent for PIH.