Transgenic Model Research Articles

Bao Yuan decoction alleviates fatigue by restraining inflammation and oxidative stress via the AMPK/CRY2/PER1 signaling pathway

Ethnopharmacological relevanceBaoyuan Decoction (BYD) was initially recorded in the classic of “Bo Ai Xin Jian” in the Ming dynasty. It is traditionally used for treating weakness and cowardice, and deficiency of vital energy. In researches related to anti-fatigue effects, the reciprocal regulation of AMPK and circadian clocks likely plays an important role in anti-fatigue mechanism, while it has not yet been revealed. Therefore, we elucidated the anti-fatigue mechanism of BYD through AMPK/CRY2/PER1 pathway. Aim of the studyTo investigate the effect and mechanism of BYD in reducing fatigue, using pharmacodynamics, network pharmacology and transcriptomics through the AMPK/CRY2/PER1 signaling pathway. Materials and methodsFirstly, the chemical constituents of BYD were qualitatively identified by UHPLC-Q-Exactive Orbitrap/MS, establishing a comprehensive strategy with an in-house library, Xcalibur software and Pubchem combined. Secondly, a Na2SO3-induced fatigue model and 2,2′-Azobis (2-methylpropionamidine) dihydrochloride (AAPH)-induced oxidative stress model were developed to evaluate the anti-fatigue and anti-oxidant activities of BYD using AB zebrafish. The anti-inflammatory activity of BYD was evaluated using CuSO4-induced and tail cutting-induced Tg (lyz: dsRed) transgenic zebrafish inflammation models. Then, target screening was performed by Swiss ADME, GeneCards, OMIM and DrugBank databases, the network was constructed using Cytoscape 3.9.0. Transcriptome and network pharmacology technology were used to investigate the related signaling pathways and potential mechanisms after treatment with BYD, which were verified by real-time quantitative PCR (RT-qPCR). ResultsIn total, 114 compounds from the water extract of BYD were identified as major compounds. Na₂SO₃-induced fatigue model and AAPH-induced oxidative stress model indicated that BYD has significant anti-fatigue and antioxidant effects. Meanwhile, BYD showed significant anti-inflammatory effects on CuSO4-induced and tail cutting-induced zebrafish inflammation models. The KEGG result of network pharmacology showed that the anti-fatigue function of BYD was mainly effected through AMPK signaling pathway. Besides, transcriptome analysis indicated that the circadian rhythm, AMPK and IL-17 signaling pathways were recommended as the main pathways related to the anti-fatigue effect of BYD. The RT-qPCR results showed that compared with a model control group, the treatment of BYD significantly elevated the expression mRNA of AMPK, CRY2 and PER1. ConclusionHerein, we identified 114 chemical constituents of BYD, performed zebrafish activity validation, while demonstrated that BYD can relieve fatigue by AMPK/CRY2/PER1 signaling pathway through network pharmacology and transcriptome.

Inhibition of miR-25 ameliorates cardiac and skeletal muscle dysfunction in aged mdx/utrn haploinsufficient (+/−) mice

Dystrophic cardiomyopathy is a significant feature of Duchenne muscular dystrophy (DMD). Increased cardiomyocyte cytosolic calcium (Ca2+) and interstitial fibrosis are major pathophysiological hallmarks that ultimately result in cardiac dysfunction. MicroRNA-25 (miR-25) has been identified as a suppressor of both sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) and mothers against decapentaplegic homolog-7 (Smad7) proteins. In this study, we created a gene transfer using an miR-25 tough decoy (TuD) RNA inhibitor delivered via recombinant adeno-associated virus serotype 9 (AAV9) to evaluate the effect of miR-25 inhibition on cardiac and skeletal muscle function in aged dystrophin/utrophin haploinsufficient mice mdx/utrn (+/-), a validated transgenic murine model of DMD. We found that the intravenous delivery of AAV9 miR-25 TuD resulted in strong and stable inhibition of cardiac miR-25 levels, together with the restoration of SERCA2a and Smad7 expression. This was associated with the amelioration of cardiomyocyte interstitial fibrosis as well as recovered cardiac function. Furthermore, the direct quadricep intramuscular injection of AAV9 miR-25 TuD significantly restored skeletal muscle Smad7 expression, reduced tissue fibrosis, and enhanced skeletal muscle performance in mdx/utrn (+/-) mice. These results imply that miR-25 TuD gene transfer may be a novel therapeutic approach to restore cardiomyocyte Ca2+ homeostasis and abrogate tissue fibrosis in DMD.

Neuroprotective effects of niclosamide on disease progression via inflammatory pathways modulation in SOD1-G93A and FUS-associated amyotrophic lateral sclerosis models

Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disease influenced by genetic, epigenetic, and environmental factors, resulting in dysfunction in cellular and molecular pathways. The limited efficacy of current treatments highlights the need for combination therapies targeting multiple aspects of the disease. Niclosamide, an anthelminthic drug listed as an essential medicine, has been repurposed in clinical trials for different diseases due to its anti-inflammatory and anti-fibrotic properties. Niclosamide can inhibit various molecular pathways (e.g., STAT3, mTOR) that are dysregulated in ALS, suggesting its potential to disrupt these altered mechanisms associated with the pathology. We administered niclosamide intraperitoneally to two transgenic murine models, SOD1-G93A and FUS mice, mimicking key pathological processes of ALS. The treatment was initiated at the onset of symptoms, and we assessed disease progression by neurological scores, rotarod and wire tests, and monitored survival. Furthermore, we investigated cellular and molecular mechanisms affected by niclosamide in the spinal cord and muscle of ALS mice. In both models, the administration of niclosamide resulted in a slowdown of disease progression, an increase in survival rates, and an improvement in tissue pathology. This was characterised by reduced gliosis, motor neuron loss, muscle atrophy, and inflammatory pathways. Based on these results, our findings demonstrate that niclosamide can impact multiple pathways involved in ALS. This multi-targeted approach leads to a slowdown in the progression of the disease, positioning niclosamide as a promising candidate for repurposing in the treatment of ALS.

Insights into the Multifaceted Roles of Thioredoxin-1 System: Exploring Knockout Murine Models.

Redox balance is increasingly identified as a major player in cellular signaling. A fundamentally simple reaction of oxidation and reduction of cysteine residues in cellular proteins is the central concept in this complex regulatory mode of protein function. Oxidation of key cysteine residues occurs at the physiological levels of reactive oxygen species (ROS), but they are reduced by a supply of thiol antioxidant molecules including glutathione, glutaredoxin, and thioredoxin. While these molecules show complex compensatory roles in experimental conditions, transgenic animal models provide a comprehensive picture to pinpoint the role of each antioxidant. In this review, we have specifically focused on the available literature on thioredoxin-1 system transgenic models that include thioredoxin and thioredoxin reductase proteins. As the identification of thioredoxin protein targets is technically challenging, the true contribution of this system in maintaining cellular balance remains unidentified, including the role of this system in the brain.

Linoleic acid exhibits anti-proliferative and anti-invasive activities in endometrial cancer cells and a transgenic model of endometrial cancer

ABSTRACT Emerging evidence has provided considerable insights into the integral function of reprogramming fatty acid metabolism in the carcinogenesis and progression of endometrial cancer. Linoleic acid, an essential fatty acid with the highest consumption in the Western diet regimen, has shown pro-tumorigenic or anti-tumorigenic effects on tumor cell growth and invasion in multiple types of cancer. However, the biological role of linoleic acid in endometrial cancer remains unclear. In the present study, we aimed to investigate the functional impact of linoleic acid on cell proliferation, invasion, and tumor growth in endometrial cancer cells and in a transgenic mouse model of endometrial cancer. The results showed that Linoleic acid significantly inhibited the proliferation of endometrial cancer cells in a dose-dependent manner. The treatment of HEC-1A and KLE cells with linoleic acid effectively increased intracellular reactive oxygen species (ROS) production, decreased mitochondrial membrane potential, caused cell cycle G1 arrest, and induced intrinsic and extrinsic apoptosis pathways. The anti-invasive ability of linoleic acid was found to be associated with the epithelial-mesenchymal transition process in both cell lines, including the decreased expression of N-cadherin, snail, and vimentin. Furthermore, treatment of Lkb1 fl/fl p53 fl/fl transgenic mice with linoleic acid for four weeks significantly reduced the growth of endometrial tumors and decreased the expression of VEGF, vimentin, Ki67, and cyclin D1 in tumor tissues. Our findings demonstrate that linoleic acid exhibits anti-proliferative and anti-invasive activities in endometrial cancer cell lines and the Lkb1 fl/fl p53 fl/fl mouse model of endometrial cancer, thus providing a pre-clinical basis for future dietary interventions with linoleic acid in endometrial cancer.

Multifunctional Liposomes Targeting Amyloid-β Oligomers for Early Diagnosis and Therapy of Alzheimer's Disease.

Early detection and treatment are crucial for Alzheimer's disease (AD) management. Current diagnostic and therapeutic methods focus on late-stage amyloid fibrils and plaques, overlooking toxic soluble amyloid β oligomers (AβOs) accumulating early in AD. A multifunctional liposome-based platform is designed for early diagnosis and therapy of AD, leveraging a novel self-assembled cyclic d,l-α-peptide (CP-2) that selectively targets AβOs. Biocompatible CP-2 conjugated liposomes (CP-2-LPs) effectively disrupt Aβ aggregation and mitigate Aβ-mediated toxicity in human neuroblastoma cells. In transgenic Caenorhabditis elegans AD models, CP-2-LPs significantly outperformed free CP-2 by improving cognitive and behavioral functions, extending lifespan, and reducing toxic AβO levels. Intravenous injection of fluorescently labeled CP-2-LPs reveals effective blood-brain barrier penetration, with significantly higher brain fluorescence in transgenic mice than WT, enabling precise diagnosis. These findings underscore CP-2-LPs as a valuable tool for early detection and targeted therapy in AD.

On the Inadequacy of the Current Transgenic Animal Models of Alzheimer's Disease: The Path Forward.

For at least two reasons, the current transgenic animal models of Alzheimer's disease (AD) appear to be patently inadequate. They may be useful in many respects, the AD models; however, they are not. First, they are incapable of developing the full spectrum of the AD pathology. Second, they respond spectacularly well to drugs that are completely ineffective in the treatment of symptomatic AD. These observations indicate that both the transgenic animal models and the drugs faithfully reflect the theory that guided the design and development of both, the amyloid cascade hypothesis (ACH), and that both are inadequate because their underlying theory is. This conclusion necessitated the formulation of a new, all-encompassing theory of conventional AD-the ACH2.0. The two principal attributes of the ACH2.0 are the following. One, in conventional AD, the agent that causes the disease and drives its pathology is the intraneuronal amyloid-β (iAβ) produced in two distinctly different pathways. Two, following the commencement of AD, the bulk of Aβ is generated independently of Aβ protein precursor (AβPP) and is retained inside the neuron as iAβ. Within the framework of the ACH2.0, AβPP-derived iAβ accumulates physiologically in a lifelong process. It cannot reach levels required to support the progression of AD; it does, however, cause the disease. Indeed, conventional AD occurs if and when the levels of AβPP-derived iAβ cross the critical threshold, elicit the neuronal integrated stress response (ISR), and trigger the activation of the AβPP-independent iAβ generation pathway; the disease commences only when this pathway is operational. The iAβ produced in this pathway reaches levels sufficient to drive the AD pathology; it also propagates its own production and thus sustains the activity of the pathway and perpetuates its operation. The present study analyzes the reason underlying the evident inadequacy of the current transgenic animal models of AD. It concludes that they model, in fact, not Alzheimer's disease but rather the effects of the neuronal ISR sustained by AβPP-derived iAβ, that this is due to the lack of the operational AβPP-independent iAβ production pathway, and that this mechanism must be incorporated into any successful AD model faithfully emulating the disease. The study dissects the plausible molecular mechanisms of the AβPP-independent iAβ production and the pathways leading to their activation, and introduces the concept of conventional versus unconventional Alzheimer's disease. It also proposes the path forward, posits the principles of design of productive transgenic animal models of the disease, and describes the molecular details of their construction.

Invertebrate genetic models of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a common adult-onset neurodegenerative disease characterized by the progressive death of motor neurons in the cerebral cortex, brain stem, and spinal cord. The exact mechanisms underlying the pathogenesis of ALS remain unclear. The current consensus regarding the pathogenesis of ALS suggests that the interaction between genetic susceptibility and harmful environmental factors is a promising cause of ALS onset. The investigation of putative harmful environmental factors has been the subject of several ongoing studies, but the use of transgenic animal models to study ALS has provided valuable information on the onset of ALS. Here, we review the current common invertebrate genetic models used to study the pathology, pathophysiology, and pathogenesis of ALS. The considerations of the usage, advantages, disadvantages, costs, and availability of each invertebrate model will also be discussed.

Adeno-associated virus-based approach for genetic modification of cardiac fibroblasts in adult rat hearts.

Cardiac fibroblasts (CFs) are an attractive target for reducing pathological cardiac remodeling, and understanding the underlying mechanisms of these processes is the key to develop successful therapies for treating the pressure-overloaded heart. CF-specific knockout (KO) mouse lines with a Cre recombinase under the control of human TCF21 (hTCF21) promoter and/or an adeno-associated virus serotype 9 (AAV9)-hTCF21 system provide a powerful tool for understanding CF biology invivo. Although a variety of rat disease models are vital for the research of cardiac fibrosis similar to mouse models, there are few rat models that employ cardiac cell-specific conditional gene modification, which has hindered the development and translational relevance of cardiac disease models. In addition, to date, there are no reports of gene manipulation specifically in rat CFs invivo. Here, we report a simplified CF-specific rat transgenic model using an AAV9-hTCF21 system that achieved a CF-specific expression of transgene in adult rat hearts. Moreover, we successfully applied this approach to specifically manipulate mitochondrial morphology in quiescent CFs. In summary, this model will allow us to develop fast and simple rat CF-specific transgenic models for studying cardiovascular diseases invivo.

Hexaconazole induces developmental toxicities via apoptosis, inflammation, and alterations of Akt and MAPK signaling cascades

Hexaconazole is a highly effective triazole fungicide that is frequently applied in various countries to elevate crop productivity. Given its long half-life and high water solubility, this fungicide is frequently detected in the environment, including water sources. Moreover, hexaconazole exerts hazardous effects on nontarget organisms. However, little is known about the toxic effects of hexaconazole on animal development. Thus, this study aimed to investigate the developmental toxicity of hexaconazole to zebrafish, a valuable animal model for toxicological studies, and elucidate the underlying mechanisms. Results showed that hexaconazole affected the viability and hatching rate of zebrafish at 96 h postfertilization. Hexaconazole-treated zebrafish showed phenotypic defects, such as reduced size of head and eyes and enlarged pericardiac edema. Moreover, hexaconazole induced apoptosis, DNA fragmentation, and inflammation in developing zebrafish. Various organ defects, including neurotoxicity, cardiovascular toxicity, and hepatotoxicity, were observed in transgenic zebrafish models olig2:dsRed, fli1:eGFP, and l-fabp:dsRed. Furthermore, hexaconazole treatment altered the Akt and MAPK signaling pathways, which possibly triggered the organ defects and other toxic mechanisms. This study demonstrated the developmental toxicity of hexaconazole to zebrafish and elucidated the underlying mechanisms.

Integrin-αvβ6 targeted peptide-toxin therapy in a novel αvβ6-expressing immunocompetent model of pancreatic cancer

Previously we reported that a novel αvβ6-specific peptide-drug conjugate (SG3299) could eliminate established human pancreatic ductal adenocarcinoma (PDAC) xenografts. However the development of effective therapies for PDAC, which is an essential need, must show efficacy in relevant immunocompetent animals. Previously we reported that the KPC mouse transgenic PDAC model that closely recapitulates most stages of development of human PDAC, unlike in humans, failed to express αvβ6 on their tumours or metastases. In this study we have taken the KPC-derived PDAC line TB32043 and engineered a variant line (TB32043mb6S2) that expresses mouse integrin αvβ6. We report that orthotopic implantation of the αvβ6 over-expressing TB32043mb6S2 cells promotes shorter overall survival and increase in metastases. Moreover, systemic treatment of mice with established TB32043mb6S2 tumours in the pancreas with SG2399 lived significantly longer (p < 0.001; mean OS 48d) compared with PBS or control SG3511 (mean OS 25.5d and 26d, respectively). Thus SG3299 is confirmed as a promising candidate therapeutic for the therapy of PDAC.

Targeting TNF/IL-17/MAPK pathway in hE2A-PBX1 leukemia: effects of OUL35, KJ-Pyr-9, and CID44216842.

t(1;19)(q23;p13) is one of the most common translocation genes in childhood acute lymphoblastic leukemia (ALL) and is also present in acute myeloid leukemia (AML) and mixed-phenotype acute leukemia (MPAL). This translocation results in the formation of the oncogenic E2A-PBX1 fusion protein, which contains a trans-activating domain from E2A and a DNA-binding homologous domain from PBX1. Despite its clear oncogenic potential, the pathogenesis of E2A-PBX1 fusion protein is not fully understood (especially in leukemias other than ALL), and effective targeted clinical therapies have not been developed. To address this, we established a stable and heritable zebrafish line expressing human E2A-PBX1 (hE2A-PBX1) for high-throughput drug screening. Blood phenotype analysis showed that hE2A-PBX1 expression induced myeloid hyperplasia by increasing myeloid differentiation propensity of hematopoietic stem cells (HSPC) and myeloid proliferation in larvae, and progressed to AML in adults. Mechanistic studies revealed that hE2A-PBX1 activated the TNF/IL-17/MAPK signaling pathway in blood cells and induced myeloid hyperplasia by upregulating the expression of runx1. Interestingly, through high-throughput drug screening, three small molecules targeting the TNF/IL-17/MAPK signaling pathway were identified, including OUL35, KJ-Pyr-9, and CID44216842, which not only alleviated the hE2A-PBX1-induced myeloid hyperplasia in zebrafish but also inhibited the growth and oncogenicity of human pre-B ALL cells with E2A-PBX1. Overall, this study provides a novel hE2APBX1 transgenic zebrafish leukemia model and identifies potential targeted therapeutic drugs, which may offer new insights into the treatment of E2A-PBX1 leukemia.

Abnormal Alpha-Synuclein Aggregates Cause Synaptic- and Microcircuit-specific Deficits in the Retinal Rod Pathway

Alpha-synuclein (α-Syn) is a key determinator of Parkinson’s disease (PD) pathology, but synapse and microcircuit pathologies in the retina underlying visual dysfunction are poorly understood. Using the old transgenic M83 PD model (16-18 monthes) coupled with histochemical and ultra-structural analyses and ophthalmologic measurements, we revealed that abnormal α-Syn aggregation in the outer plexiform layer (OPL) was associated with degeneration in the CtBP2+ ribbon synapses of photoreceptor terminals and PKCα+ rod bipolar cells terminals while α-Syn aggregates in the inner retina correlated with the reduction and degeneration of tyrosine hydroxylase (TH)- and parvalbumin (PV)-positive amacrine cells. Phosphorylated Ser129 α-synuclein expression was strikingly restricted in the OPL with the most severe degenerations in the entire retina, including mitochondrial degeneration and loss of ribbon synapses in 16-18-month-old mice. These synapse- and microcircuit-specific deficits of the rod pathway at the CtBP2+ rod terminals and PKCα+ rod bipolar and amacrine cells were associated with attenuated a- and b-wave amplitudes and oscillatory potentials on the electroretinogram. They were also associated with the impairment of visual functions, including reduced contrast sensitivity and impairment of the middle range of spatial frequencies. Collectively, these findings demonstrate that α-Syn aggregates cause the synapse- and microcircuit-specific deficits of the rod pathway and the most severe damage to the OPL, providing the retinal synaptic and microcircuit basis for visual dysfunctions in PD.

The new era of cardiovascular research: revolutionizing cardiovascular research with 3D models in a dish.

Cardiovascular research has heavily relied on studies using patient samples and animal models. However, patient studies often miss the data from the crucial early stage of cardiovascular diseases, as obtaining primary tissues at this stage is impracticable. Transgenic animal models can offer some insights into disease mechanisms, although they usually do not fully recapitulate the phenotype of cardiovascular diseases and their progression. In recent years, a promising breakthrough has emerged in the form of invitro three-dimensional (3D) cardiovascular models utilizing human pluripotent stem cells. These innovative models recreate the intricate 3D structure of the human heart and vessels within a controlled environment. This advancement is pivotal as it addresses the existing gaps in cardiovascular research, allowing scientists to study different stages of cardiovascular diseases and specific drug responses using human-origin models. In this review, we first outline various approaches employed to generate these models. We then comprehensively discuss their applications in studying cardiovascular diseases by providing insights into molecular and cellular changes associated with cardiovascular conditions. Moreover, we highlight the potential of these 3D models serving as a platform for drug testing to assess drug efficacy and safety. Despite their immense potential, challenges persist, particularly in maintaining the complex structure of 3D heart and vessel models and ensuring their function is comparable to real organs. However, overcoming these challenges could revolutionize cardiovascular research. It has the potential to offer comprehensive mechanistic insights into human-specific disease processes, ultimately expediting the development of personalized therapies.

An antitumor arabinan from Glehnia littoralis activates immunity and inhibits angiogenesis

Glehnia littoralis is an edible plant with significant medicinal value. To further elucidate the potential functional components for developing antitumor agents or functional foods, the polysaccharides in this plant were investigated, and a homogeneous polysaccharide, GLP90-2, was obtained through extraction and ethanol precipitation. By employing methylation, GC–MS, FT-IR, and NMR analysis, GLP90-2 was identified as an arabinan having a molecular weight of 7.76 × 103 g/mol and consisting of three types of residues: α-l-Araf-(1→, →5)-α-l-Araf-(1→, and →3,5)-α-l-Araf-(1→. The subsequent functional analysis revealed that GLP90-2 suppressed tumor development and metastasis in a zebrafish model. Mechanistic studies have shown that GLP90-2 promoted the maturation of DC2.4 cells and macrophages and enhanced the expression of immune-related cytokines, which may be attributed to the interaction between GLP90-2 and TLR-4. Additionally, GLP90-2 exhibited a strong interaction with PD-1, contributing to the activation of immunity. Furthermore, GLP90-2 suppressed angiogenesis in the transgenic zebrafish model, and this impact may be ascribed to the modulation of the VEGF/VEGFR-2 signaling pathway. All the results indicate that GLP90-2 demonstrates a strong tumor immunotherapy effect in vivo and has high potential for development.

Synergistic Anticancer Activity of Plumbagin and Xanthohumol Combination on Pancreatic Cancer Models.

Among diverse cancers, pancreatic cancer is one of the most aggressive types due to inadequate diagnostic options and treatments available. Therefore, there is a necessity to use combination chemotherapy options to overcome the chemoresistance of pancreatic cancer cells. Plumbagin and xanthohumol, natural compounds isolated from the Plumbaginaceae family and Humulus lupulus, respectively, have been used to treat various cancers. In this study, we investigated the anticancer effects of a combination of plumbagin and xanthohumol on pancreatic cancer models, as well as the underlying mechanism. We have screened in vitro numerous plant-derived extracts and compounds and tested in vivo the most effective combination, plumbagin and xanthohumol, using a transgenic model of pancreatic cancer KPC (KrasLSL.G12D/+; p53R172H/+; PdxCretg/+). A significant synergistic anticancer activity of plumbagin and xanthohumol combinations on different pancreatic cancer cell lines was found. The combination treatment of plumbagin and xanthohumol influences the levels of B-cell lymphoma (BCL2), which are known to be associated with apoptosis in both cell lysates and tissues. More importantly, the survival of a transgenic mouse model of pancreatic cancer KPC treated with a combination of plumbagin and xanthohumol was significantly increased, and the effect on BCL2 levels has been confirmed. These results provide a foundation for a potential new treatment for pancreatic cancer based on plumbagin and xanthohumol combinations.

Lentiviral Vector Preparation for Efficient Gene and MicroRNA Modulation of Peritoneal Cavity Tissue-Resident Macrophages In Vivo in Mice.

Peritoneal tissue-resident macrophages have broad functions in the maintenance of homeostasis and are involved in pathologies within local and neighboring tissues. Their functions are dictated by microenvironmental cues; thus, it is essential to investigate their behavior in an in vivo physiological niche. Currently, specific peritoneal macrophage-targeting methodologies employ whole-mouse transgenic models. Here, a protocol for effective in vivo modulation of mRNA and small RNA species (e.g., microRNA) expression in peritoneal macrophages using lentivirus particles is described. Lentivirus preparations were made in HEK293T cells and purified on a single sucrose layer. In vivo validation of lentivirus effectivity following intraperitoneal injection revealed predominant infection of macrophages restricted to local tissue. Targeting of peritoneal macrophages was successful during homeostasis and thioglycolate-induced peritonitis. The limitations of the protocol, including low-level inflammation induced by intraperitoneal delivery of lentivirus and time restrictions for potential experiments, are discussed. Overall, this study presents a quick and accessible protocol for the rapid assessment of gene function in peritoneal macrophages in vivo.

Knockdown of ANGPTL2 promotes left ventricular systolic dysfunction by upregulation of NOX4 in mice.

Background: Angiopoietin-like 2 (ANGPTL2) is a pro-inflammatory and pro-oxidant circulating protein that predicts and promotes chronic inflammatory diseases such as atherosclerosis in humans. Transgenic murine models demonstrated the deleterious role of ANGPTL2 in vascular diseases, while deletion of ANGPTL2 was protective. The nature of its role in cardiac tissues is, however, less clear. Indeed, in adult mice knocked down (KD) for ANGPTL2, we recently reported a mild left ventricular (LV) dysfunction originating from a congenital aortic valve stenosis, demonstrating that ANGPTL2 is essential to cardiac development and function. Hypothesis: Because we originally demonstrated that the KD of ANGPTL2 protected vascular endothelial function via an upregulation of arterial NOX4, promoting the beneficial production of dilatory H2O2, we tested the hypothesis that increased cardiac NOX4 could negatively affect cardiac redox and remodeling and contribute to LV dysfunction observed in adult Angptl2-KD mice. Methods and results: Cardiac expression and activity of NOX4 were higher in KD mice, promoting higher levels of cardiac H2O2 when compared to wild-type (WT) mice. Immunofluorescence showed that ANGPTL2 and NOX4 were co-expressed in cardiac cells from WT mice and both proteins co-immunoprecipitated in HEK293 cells, suggesting that ANGPTL2 and NOX4 physically interact. Pressure overload induced by transverse aortic constriction surgery (TAC) promoted LV systolic dysfunction in WT mice but did not further exacerbate the dysfunction in KD mice. Importantly, the severity of LV systolic dysfunction in KD mice (TAC and control SHAM) correlated with cardiac Nox4 expression. Injection of an adeno-associated virus (AAV9) delivering shRNA targeting cardiac Nox4 expression fully reversed LV systolic dysfunction in KD-SHAM mice, demonstrating the causal role of NOX4 in cardiac dysfunction in KD mice. Targeting cardiac Nox4 expression in KD mice also induced an antioxidant response characterized by increased expression of NRF2/KEAP1 and catalase. Conclusion: Together, these data reveal that the absence of ANGPTL2 induces an upregulation of cardiac NOX4 that contributes to oxidative stress and LV dysfunction. By interacting and repressing cardiac NOX4, ANGPTL2 could play a new beneficial role in the maintenance of cardiac redox homeostasis and function.

Human-induced pluripotent stem cell-derived neural stem/progenitor cell ex vivo gene therapy with synaptic organizer CPTX for spinal cord injury

The transplantation of neural stem/progenitor cells (NS/PCs) derived from human induced pluripotent stem cells (hiPSCs) has shown promise in spinal cord injury (SCI) model animals. Establishing a functional synaptic connection between the transplanted and host neurons is crucial for motor function recovery. To boost therapeutic outcomes, we developed an exvivo gene therapy aimed at promoting synapse formation by expressing the synthetic excitatory synapse organizer CPTX in hiPSC-NS/PCs. Using an immunocompromised transgenic rat model of SCI, we evaluated the effects of transplanting CPTX-expressing hiPSC-NS/PCs using histological and functional analyses. Our findings revealed a significant increase in excitatory synapse formation at the transplantation site. Retrograde monosynaptic tracing indicated extensive integration of transplanted neurons into the surrounding neuronal tracts facilitated by CPTX. Consequently, locomotion and spinal cord conduction significantly improved. Thus, exvivo gene therapy targeting synapse formation holds promise for future clinical applications and offers potential benefits to individuals with SCI.

SLAM-ITseq identifies that Nrf2 induces liver regeneration through the pentose phosphate pathway

The liver exhibits a remarkable capacity to regenerate following injury. Despite this unique attribute, toxic injury is a leading cause of liver failure. The temporal processes by which the liver senses injury and initiates regeneration remain unclear. Here, we developed a transgenic zebrafish model wherein hepatocyte-specific expression of uracil phosphoribosyltransferase (UPRT) enabled the implementation of SLAM-ITseq to investigate the nascent transcriptome during initiation of liver injury and regeneration. Using this approach, we identified a rapid metabolic transition from the fed to the fasted state that was followed by induction of the nuclear erythroid 2-related factor (Nrf2) antioxidant program. We find that activation of Nrf2 in hepatocytes is required to induce the pentose phosphate pathway (PPP) and improve survival following liver injury. Mechanistically, we demonstrate that inhibition of the PPP disrupts nucleotide biosynthesis to prevent liver regeneration. Together, these studies provide fundamental insights into the mechanism by which early metabolic adaptation to injury facilitates tissue regeneration.