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Background and Aims: Angiopoietin-like 3 (ANGPTL3) and 4 (ANGPTL4) inhibit lipoprotein lipase to regulate tissue fatty acid uptake from triglyceride-rich lipoproteins such as VLDL. While pharmacological inhibition of ANGPTL3 is being evaluated as lipid-lowering strategy, systemic ANGPTL4 inhibition is not pursued due to adverse effects. This study aimed to compare the therapeutic potential of liver-specific Angptl3 and Angptl4 silencing to attenuate hyperlipidemia and atherosclerosis development in APOE*3-Leiden.CETP mice, a well-established humanized model for lipoprotein metabolism., Methods and Results: Mice were subcutaneously injected twice-weekly with saline or liver-targeted antisense oligonucleotides against Angptl3, Angptl4, both, or a scrambled oligonucleotide. Plasma lipid levels, VLDL clearance and hepatic VLDL production were determined, and atherosclerosis development was assessed. For toxicological evaluation, cynomolgus monkeys were treated with three dosages of liver-targeted ANGPTL4-silencing oligonucleotides.Liver-targeted Angptl4 silencing reduced plasma triglycerides (-48%) and total cholesterol (-56%), explained by higher VLDL-derived fatty acid uptake by brown adipose tissue and lower VLDL production by the liver. Accordingly, Angptl4 silencing reduced atherosclerotic lesion size (-86%) and improved lesion stability. Hepatic Angptl3 silencing similarly attenuated hyperlipidemia and atherosclerosis development. While Angptl3 and Angptl4 silencing lowered plasma triglycerides in the refed and fasted state, respectively, combined Angptl3/4 silencing lowered plasma triglycerides independent of nutritional state. In cynomolgus monkeys, anti-ANGPTL4 ASO treatment was well tolerated without adverse effects., Conclusions: Liver-targeted Angptl4 silencing potently attenuates hyperlipidemia and atherosclerosis development in APOE*3-Leiden.CETP mice, and liver-targeted ANGPTL4 silencing is well-tolerated in non-human primates. These data warrant further clinical development of liver-targeted ANGPTL4 silencing., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology.)

A mechanistic connection between aging and development is largely unexplored. Through profiling age-related chromatin and transcriptional changes across 22 murine cell types, analyzed alongside previous mouse and human organismal maturation datasets, we uncovered a transcription factor binding site (TFBS) signature common to both processes. Early-life candidate cis-regulatory elements (cCREs), progressively losing accessibility during maturation and aging, are enriched for cell-type identity TFBSs. Conversely, cCREs gaining accessibility throughout life have a lower abundance of cell identity TFBSs but elevated activator protein 1 (AP-1) levels. We implicate TF redistribution toward these AP-1 TFBS-rich cCREs, in synergy with mild downregulation of cell identity TFs, as driving early-life cCRE accessibility loss and altering developmental and metabolic gene expression. Such remodeling can be triggered by elevating AP-1 or depleting repressive H3K27me3. We propose that AP-1-linked chromatin opening drives organismal maturation by disrupting cell identity TFBS-rich cCREs, thereby reprogramming transcriptome and cell function, a mechanism hijacked in aging through ongoing chromatin opening., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Aims: APOC3, ANGPTL3, and ANGPTL4 are circulating proteins that are actively pursued as pharmacological targets to treat dyslipidaemia and reduce the risk of atherosclerotic cardiovascular disease. Here, we used human genetic data to compare the predicted therapeutic and adverse effects of APOC3, ANGPTL3, and ANGPTL4 inactivation., Methods and Results: We conducted drug-target Mendelian randomization analyses using variants in proximity to the genes associated with circulating protein levels to compare APOC3, ANGPTL3, and ANGPTL4 as drug targets. We obtained exposure and outcome data from large-scale genome-wide association studies and used generalized least squares to correct for linkage disequilibrium-related correlation. We evaluated five primary cardiometabolic endpoints and screened for potential side effects across 694 disease-related endpoints, 43 clinical laboratory tests, and 11 internal organ MRI measurements. Genetically lowering circulating ANGPTL4 levels reduced the odds of coronary artery disease (CAD) [odds ratio, 0.57 per s.d. protein (95% CI 0.47-0.70)] and Type 2 diabetes (T2D) [odds ratio, 0.73 per s.d. protein (95% CI 0.57-0.94)]. Genetically lowering circulating APOC3 levels also reduced the odds of CAD [odds ratio, 0.90 per s.d. protein (95% CI 0.82-0.99)]. Genetically lowered ANGPTL3 levels via common variants were not associated with CAD. However, meta-analysis of protein-truncating variants revealed that ANGPTL3 inactivation protected against CAD (odds ratio, 0.71 per allele [95%CI, 0.58-0.85]). Analysis of lowered ANGPTL3, ANGPTL4, and APOC3 levels did not identify important safety concerns., Conclusion: Human genetic evidence suggests that therapies aimed at reducing circulating levels of ANGPTL3, ANGPTL4, and APOC3 reduce the risk of CAD. ANGPTL4 lowering may also reduce the risk of T2D., Competing Interests: Conflict of interest: F.L. is a part-time employee of Lipigon Pharmaceuticals AB. S.K.N. is the chief executive officer of Lipigon. S.K. is a paid consultant for Lipigon., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Competing Interests: Conflict of Interest Disclosure The authors do not have any conflicts of interest to declare in relation to this work.

Hematopoiesis produces diverse blood cell lineages to meet the basal needs and sudden demands of injury or infection. A rapid response to such challenges requires the expansion of specific lineages and a prompt return to balanced steady-state levels, necessitating tightly coordinated regulation. Previously we identified a requirement for the zinc finger and broad complex, tramtrak, bric-a-brac domain-containing 11 (ZBTB11) transcription factor in definitive hematopoiesis using a forward genetic screen for zebrafish myeloid mutants. To understand its relevance to mammalian systems, we extended these studies to mice. When Zbtb11 was deleted in the hematopoietic compartment, embryos died at embryonic day (E) 18.5 with hematopoietic failure. Zbtb11 hematopoietic knockout (Zbtb11hKO) hematopoietic stem cells (HSCs) were overabundantly specified from E14.5 to E17.5 compared with those in controls. Overspecification was accompanied by loss of stemness, inability to differentiate into committed progenitors and mature lineages in the fetal liver, failure to seed fetal bone marrow, and total hematopoietic failure. The Zbtb11hKO HSCs did not proliferate in vitro and were constrained in cell cycle progression, demonstrating the cell-intrinsic role of Zbtb11 in proliferation and cell cycle regulation in mammalian HSCs. Single-cell RNA sequencing analysis identified that Zbtb11-deficient HSCs were underrepresented in an erythroid-primed subpopulation and showed downregulation of oxidative phosphorylation pathways and dysregulation of genes associated with the hematopoietic niche. We identified a cell-intrinsic requirement for Zbtb11-mediated gene regulatory networks in sustaining a pool of maturation-capable HSCs and progenitor cells., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Background: There is great interest to engineer in vitro models that allow the study of complex biological processes of the microvasculature with high spatiotemporal resolution. Microfluidic systems are currently used to engineer microvasculature in vitro, which consists of perfusable microvascular networks (MVNs). These are formed through spontaneous vasculogenesis and exhibit the closest resemblance to physiological microvasculature. Unfortunately, under standard culture conditions and in the absence of co-culture with auxiliary cells as well as protease inhibitors, pure MVNs suffer from a short-lived stability., Methods: Herein, we introduce a strategy for stabilization of MVNs through macromolecular crowding (MMC) based on a previously established mixture of Ficoll macromolecules. The biophysical principle of MMC is based on macromolecules occupying space, thus increasing the effective concentration of other components and thereby accelerating various biological processes, such as extracellular matrix deposition. We thus hypothesized that MMC will promote the accumulation of vascular ECM (basement membrane) components and lead to a stabilization of MVN with improved functionality., Results: MMC promoted the enrichment of cellular junctions and basement membrane components, while reducing cellular contractility. The resulting advantageous balance of adhesive forces over cellular tension resulted in a significant stabilization of MVNs over time, as well as improved vascular barrier function, closely resembling that of in vivo microvasculature., Conclusion: Application of MMC to MVNs in microfluidic devices provides a reliable, flexible and versatile approach to stabilize engineered microvessels under simulated physiological conditions., (© 2023. The Author(s).)

Megakaryocytes (MK) generate platelets. Recently, we and others, have reported MK also regulate hematopoietic stem cells (HSC). Here we show high ploidy large cytoplasmic megakaryocytes (LCM) are critical negative regulators of HSC and critical for platelet formation. Using a mouse knockout model (Pf4-Srsf3 Δ/Δ ) with normal MK numbers, but essentially devoid of LCM, we demonstrate a pronounced increase in BM HSC concurrent with endogenous mobilization and extramedullary hematopoiesis. Severe thrombocytopenia is observed in animals with diminished LCM, although there is no change in MK ploidy distribution, uncoupling endoreduplication and platelet production. When HSC isolated from a microenvironment essentially devoid of LCM reconstitute hematopoiesis in lethally irradiated mice, the absence of LCM increases HSC in BM, blood and spleen, and the recapitulation of thrombocytopenia. In contrast, following a competitive transplant using minimal numbers of WT HSC together with HSC from a microenvironment with diminished LCM, sufficient WT HSC-generated LCM regulates a normal HSC pool and prevents thrombocytopenia. Importantly, LCM are conserved in humans., (© 2023. Crown.)

Angiopoietin-like 4 (ANGPTL4) is an important regulator of plasma triglyceride (TG) levels and an attractive pharmacological target for lowering plasma lipids and reducing cardiovascular risk. Here, we aimed to study the efficacy and safety of silencing ANGPTL4 in the livers of mice using hepatocyte-targeting GalNAc-conjugated antisense oligonucleotides (ASOs). Compared with injections with negative control ASO, four injections of two different doses of ANGPTL4 ASO over 2 weeks markedly downregulated ANGPTL4 levels in liver and adipose tissue, which was associated with significantly higher adipose LPL activity and lower plasma TGs in fed and fasted mice, as well as lower plasma glucose levels in fed mice. In separate experiments, injection of two different doses of ANGPTL4 ASO over 20 weeks of high-fat feeding reduced hepatic and adipose ANGPTL4 levels but did not trigger mesenteric lymphadenopathy, an acute phase response, chylous ascites, or any other pathological phenotypes. Compared with mice injected with negative control ASO, mice injected with ANGPTL4 ASO showed reduced food intake, reduced weight gain, and improved glucose tolerance. In addition, they exhibited lower plasma TGs, total cholesterol, LDL-C, glucose, serum amyloid A, and liver TG levels. By contrast, no significant difference in plasma alanine aminotransferase activity was observed. Overall, these data suggest that ASOs targeting ANGPTL4 effectively reduce plasma TG levels in mice without raising major safety concerns., Competing Interests: Conflict of interest A. S., S. M., and F. J. are employees of Secarna Pharmaceuticals. E. K., M. M. J., and S. K. N. are employees of Lipigon Pharmaceuticals. S. K. is a paid consultant for Lipigon. All other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The impact of aging on intestinal stem cells (ISCs) has not been fully elucidated. In this study, we identified widespread epigenetic and transcriptional alterations in old ISCs. Using a reprogramming algorithm, we identified a set of key transcription factors (Egr1, Irf1, FosB) that drives molecular and functional differences between old and young states. Overall, by dissecting the molecular signature of aged ISCs, our study identified transcription factors that enhance the regenerative capacity of ISCs., (© 2022. The Author(s).)

Cold-induced activation of brown adipose tissue (BAT) has an important impact on systemic lipoprotein metabolism by accelerating the processing of circulating triglyceride-rich lipoproteins (TRL). Lipoprotein lipase (LPL) expressed by adipocytes is translocated via endothelial to the capillary lumen, where LPL acts as the central enzyme for the vascular lipoprotein processing. Based on preliminary data showing that LPL is not only expressed in adipocytes but also in endothelial cells of cold-activated BAT, we aimed to dissect the relevance of endothelial versus adipocyte LPL for lipid and energy metabolism in the context of adaptive thermogenesis. By metabolic studies we found that cold-induced triglyceride uptake into BAT, lipoprotein disposal, glucose uptake and adaptive thermogenesis were not impaired in mice lacking Lpl exclusively in endothelial cells. This finding may be explained by a compensatory upregulation in the expression of adipocyte-derived Lpl and endothelial lipase (Lipg) ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Thiemann, Schwaerzer, Evangelakos, Fuh, Jaeckstein, Behrens, Nilsson, Kumari, Scheja, Pfeifer, Heeren and Heine.)

RNA processing is increasingly recognized as a critical control point in the regulation of different hematopoietic lineages including megakaryocytes responsible for the production of platelets. Platelets are anucleate cytoplasts that contain a rich repertoire of RNAs encoding proteins with essential platelet functions derived from the parent megakaryocyte. It is largely unknown how RNA binding proteins contribute to the development and functions of megakaryocytes and platelets. We show that serine-arginine-rich splicing factor 3 (SRSF3) is essential for megakaryocyte maturation and generation of functional platelets. Megakaryocyte-specific deletion of Srsf3 in mice led to macrothrombocytopenia characterized by megakaryocyte maturation arrest, dramatically reduced platelet counts, and abnormally large functionally compromised platelets. SRSF3 deficient megakaryocytes failed to reprogram their transcriptome during maturation and to load platelets with RNAs required for normal platelet function. SRSF3 depletion led to nuclear accumulation of megakaryocyte mRNAs, demonstrating that SRSF3 deploys similar RNA regulatory mechanisms in megakaryocytes as in other cell types. Our study further suggests that SRSF3 plays a role in sorting cytoplasmic megakaryocyte RNAs into platelets and demonstrates how SRSF3-mediated RNA processing forms a central part of megakaryocyte gene regulation. Understanding SRSF3 functions in megakaryocytes and platelets provides key insights into normal thrombopoiesis and platelet pathologies as SRSF3 RNA targets in megakaryocytes are associated with platelet diseases., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

The development of bone-like tissues in vitro that exhibit key features similar to those in vivo is needed to produce tissue models for drug screening and the study of bone physiology and disease pathogenesis. Extracellular matrix (ECM) is a predominant component of bone in vivo; however, as ECM assembly is sub-optimal in vitro, current bone tissue engineering approaches are limited by an imbalance in ECM-to-cell ratio. We amplified the deposition of osteoblastic ECM by supplementing dextran sulfate (DxS) into osteogenically induced cultures of human mesenchymal stem cells (MSCs). DxS, previously implicated to act as a macromolecular crowder, was recently demonstrated to aggregate and co-precipitate major ECM components, including collagen type I, thereby amplifying its deposition. This effect was re-confirmed for MSC cultures undergoing osteogenic induction, where DxS supplementation augmented collagen type I deposition, accompanied by extracellular osteocalcin accumulation. The resulting differentiated osteoblasts exhibited a more mature osteogenic gene expression profile, indicated by a strong upregulation of the intermediate and late osteogenic markers ALP and OCN, respectively. The associated cellular microenvironment was also enriched in bone morphogenetic protein 2 (BMP-2). Interestingly, the resulting decellularized matrices exhibited the strongest osteo-inductive effects on re-seeded MSCs, promoted cell proliferation, osteogenic marker expression and ECM calcification. Taken together, these findings suggest that DxS-mediated enhancement of osteogenic differentiation by MSCs is mediated by the amplified ECM, which is enriched in osteo-inductive factors. We have thus established a simple and reproducible approach to generate ECM-rich bone-like tissue in vitro with sequestration of osteo-inductive factors. STATEMENT OF SIGNIFICANCE: As extracellular matrix (ECM) assembly is significantly retarded in vitro, the imbalance in ECM-to-cell ratio hampers current in vitro bone tissue engineering approaches in their ability to faithfully resemble their in vivo counterpart. We addressed this limitation by leveraging a poly-electrolyte mediated co-assembly and amplified deposition of ECM during osteogenic differentiation of human mesenchymal stem cells (MSCs). The resulting pericelluar space in culture was enriched in organic and inorganic bone ECM components, as well as osteo-inductive factors, which promoted the differentiation of MSCs towards a more mature osteoblastic phenotype. These findings thus demonstrated a simple and reproducible approach to generate ECM-rich bone-like tissue in vitro with a closer recapitulation of the in vivo tissue niche., Competing Interests: Declaration of Competing Interest There are no conflicts to declare., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Understanding the spatial gene expression and regulation in the heart is key to uncovering its developmental and physiological processes, during homeostasis and disease. Numerous techniques exist to gain gene expression and regulation information in organs such as the heart, but few utilize intuitive true-to-life three-dimensional representations to analyze and visualise results. Here we combined transcriptomics with 3D-modelling to interrogate spatial gene expression in the mammalian heart. For this, we microdissected and sequenced transcriptome-wide 18 anatomical sections of the adult mouse heart. Our study has unveiled known and novel genes that display complex spatial expression in the heart sub-compartments. We have also created 3D-cardiomics, an interface for spatial transcriptome analysis and visualization that allows the easy exploration of these data in a 3D model of the heart. 3D-cardiomics is accessible from http://3d-cardiomics.erc.monash.edu/., (Copyright © 2021. Published by Elsevier Ltd.)

Astrocytes play critical roles after brain injury, but their precise function is poorly defined. Utilizing single-nuclei transcriptomics to characterize astrocytes after ischemic stroke in the visual cortex of the marmoset monkey, we observed nearly complete segregation between stroke and control astrocyte clusters. Screening for the top 30 differentially expressed genes that might limit stroke recovery, we discovered that a majority of astrocytes expressed RTN4A/ NogoA, a neurite-outgrowth inhibitory protein previously only associated with oligodendrocytes. NogoA upregulation on reactive astrocytes post-stroke was significant in both the marmoset and human brain, whereas only a marginal change was observed in mice. We determined that NogoA mediated an anti-inflammatory response which likely contributes to limiting the infiltration of peripheral macrophages into the surviving parenchyma., (© 2021. The Author(s).)

Regulatory T cell (Treg) reconstitution is essential for reestablishing tolerance and maintaining homeostasis following stem-cell transplantation. We previously reported that bone marrow (BM) is highly enriched in autophagy-dependent Treg and autophagy disruption leads to a significant Treg loss, particularly BM-Treg. To correct the known Treg deficiency observed in chronic graft-versus-host disease (cGVHD) patients, low dose IL-2 infusion has been administered, substantially increasing peripheral Treg (pTreg) numbers. However, as clinical responses were only seen in ∼50% of patients, we postulated that pTreg augmentation was more robust than for BM-Treg. We show that BM-Treg and pTreg have distinct characteristics, indicated by differential transcriptome expression for chemokine receptors, transcription factors, cell cycle control of replication and genes linked to Treg function. Further, BM-Treg were more quiescent, expressed lower FoxP3, were highly enriched for co-inhibitory markers and more profoundly depleted than splenic Treg in cGVHD mice. In vivo our data are consistent with the BM and not splenic microenvironment is, at least in part, driving this BM-Treg signature, as adoptively transferred splenic Treg that entered the BM niche acquired a BM-Treg phenotype. Analyses identified upregulated expression of IL-9R, IL-33R, and IL-7R in BM-Treg. Administration of the T cell produced cytokine IL-2 was required by splenic Treg expansion but had no impact on BM-Treg, whereas the converse was true for IL-9 administration. Plasmacytoid dendritic cells (pDCs) within the BM also may contribute to BM-Treg maintenance. Using pDC-specific BDCA2-DTR mice in which diptheria toxin administration results in global pDC depletion, we demonstrate that pDC depletion hampers BM, but not splenic, Treg homeostasis. Together, these data provide evidence that BM-Treg and splenic Treg are phenotypically and functionally distinct and influenced by niche-specific mediators that selectively support their respective Treg populations. The unique properties of BM-Treg should be considered for new therapies to reconstitute Treg and reestablish tolerance following SCT., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Nicholls, Cao, Le Texier, Xiong, Hunter, Llanes, Aguliar, Schroder, Phipps, Lynch, Cao, Heazlewood, Williams, Clouston, Nefzger, Polo, Nilsson, Blazar and MacDonald.)

With age, hematopoietic stem cells (HSC) undergo changes in function, including reduced regenerative potential and loss of quiescence, which is accompanied by a significant expansion of the stem cell pool that can lead to haematological disorders. Elevated metabolic activity has been implicated in driving the HSC ageing phenotype. Here we show that nicotinamide riboside (NR), a form of vitamin B3, restores youthful metabolic capacity by modifying mitochondrial function in multiple ways including reduced expression of nuclear encoded metabolic pathway genes, damping of mitochondrial stress and a decrease in mitochondrial mass and network-size. Metabolic restoration is dependent on continuous NR supplementation and accompanied by a shift of the aged transcriptome towards the young HSC state, more youthful bone marrow cellular composition and an improved regenerative capacity in a transplant setting. Consequently, NR administration could support healthy ageing by re-establishing a more youthful hematopoietic system.

Increased levels of apolipoprotein CIII (apoCIII), a key regulator of lipid metabolism, result in obesity-related metabolic derangements. We investigated mechanistically whether lowering or preventing high-fat diet (HFD)-induced increase in apoCIII protects against the detrimental metabolic consequences. Mice, first fed HFD for 10 weeks and thereafter also given an antisense (ASO) to lower apoCIII, already showed reduced levels of apoCIII and metabolic improvements after 4 weeks, despite maintained obesity. Prolonged ASO treatment reversed the metabolic phenotype due to increased lipase activity and receptor-mediated hepatic uptake of lipids. Fatty acids were transferred to the ketogenic pathway, and ketones were used in brown adipose tissue (BAT). This resulted in no fat accumulation and preserved morphology and function of liver and BAT. If ASO treatment started simultaneously with the HFD, mice remained lean and metabolically healthy. Thus, lowering apoCIII protects against and reverses the HFD-induced metabolic phenotype by promoting physiological insulin sensitivity., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

In response to cold exposure, thermogenic adipocytes internalize large amounts of fatty acids after lipoprotein lipase-mediated hydrolysis of triglyceride-rich lipoproteins (TRL) in the capillary lumen of brown adipose tissue (BAT) and white adipose tissue (WAT). Here, we show that in cold-exposed mice, vascular endothelial cells in adipose tissues endocytose substantial amounts of entire TRL particles. These lipoproteins subsequently follow the endosomal-lysosomal pathway, where they undergo lysosomal acid lipase (LAL)-mediated processing. Endothelial cell-specific LAL deficiency results in impaired thermogenic capacity as a consequence of reduced recruitment of brown and brite/beige adipocytes. Mechanistically, TRL processing by LAL induces proliferation of endothelial cells and adipocyte precursors via beta-oxidation-dependent production of reactive oxygen species, which in turn stimulates hypoxia-inducible factor-1α-dependent proliferative responses. In conclusion, this study demonstrates a physiological role for TRL particle uptake into BAT and WAT and establishes endothelial lipoprotein processing as an important determinant of adipose tissue remodeling during thermogenic adaptation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The reprogramming of human somatic cells to primed or naive induced pluripotent stem cells recapitulates the stages of early embryonic development 1-6 . The molecular mechanism that underpins these reprogramming processes remains largely unexplored, which impedes our understanding and limits rational improvements to reprogramming protocols. Here, to address these issues, we reconstruct molecular reprogramming trajectories of human dermal fibroblasts using single-cell transcriptomics. This revealed that reprogramming into primed and naive pluripotency follows diverging and distinct trajectories. Moreover, genome-wide analyses of accessible chromatin showed key changes in the regulatory elements of core pluripotency genes, and orchestrated global changes in chromatin accessibility over time. Integrated analysis of these datasets revealed a role for transcription factors associated with the trophectoderm lineage, and the existence of a subpopulation of cells that enter a trophectoderm-like state during reprogramming. Furthermore, this trophectoderm-like state could be captured, which enabled the derivation of induced trophoblast stem cells. Induced trophoblast stem cells are molecularly and functionally similar to trophoblast stem cells derived from human blastocysts or first-trimester placentas 7 . Our results provide a high-resolution roadmap for the transcription-factor-mediated reprogramming of human somatic cells, indicate a role for the trophectoderm-lineage-specific regulatory program during this process, and facilitate the direct reprogramming of somatic cells into induced trophoblast stem cells.

The functional group tolerance and simplicity of reversible addition fragmentation chain transfer (RAFT) polymerization enable its use in the preparation of a wide range of functional polymer architectures for a variety of applications, including drug delivery. Given the role of tumor-associated macrophages (TAMs) in cancer and their dependence on the tyrosine kinase receptor FMS (CSF-1R), the key aim of this work was to achieve effective delivery of an FMS inhibitor to cells using a polymer delivery system. Such a system has the potential to exploit biological features specific to macrophages and therefore provide enhanced selectivity. Building on our prior work, we have prepared RAFT polymers based on a poly(butyl methacrylate- co -methacrylic acid) diblock, which were extended with a hydrophilic block, a cross-linker, and a mannose-based monomer scaffold, exploiting the abundance of macrophage mannose receptors (MMRs, CD206) on the surface of macrophages. We demonstrate that the prepared polymers can be assembled into nanoparticles and are successfully internalized into macrophages, in part, via the MMR (CD206). Finally, we showcase the developed nanoparticles in the delivery of an FMS inhibitor to cells, resulting in inhibition of the FMS receptor. As such, this study lays the groundwork for further drug-delivery studies aimed at specifically targeting TAMs with molecularly targeted therapeutics.

Multiple protocols have been published for generation of iMGLs from hESCs/iPSCs. To date, there are no guides to assist researchers to determine the most appropriate methodology for microglial studies. To establish a framework to facilitate future microglial studies, we first performed a comparative transcriptional analysis between iMGLs derived using three published datasets, which allowed us to establish the baseline protocol that is most representative of bona fide human microglia. Secondly, using CRISPR to tag the classic microglial marker CX3CR1 with nanoluciferase and tdTomato, we generated and functionally validated a reporter ESC line. Finally, using this cell line, we demonstrated that co-culture of iMGL precursors with human glia and neurons enhanced transcriptional resemblance of iMGLs to ex vivo microglia. Together, our comprehensive molecular analysis and reporter cell line are a useful resource for neurobiologists seeking to use iMGLs for disease modeling and drug screening studies., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Diabetic nephropathy (DN) is the leading cause of end-stage kidney disease. TGF-β1/Smad3 signalling plays a major pathological role in DN; however, the contribution of Smad4 has not been examined. Smad4 depletion in the kidney using anti-Smad4 locked nucleic acid halted progressive podocyte damage and glomerulosclerosis in mouse type 2 DN, suggesting a pathogenic role of Smad4 in podocytes. Smad4 is upregulated in human and mouse podocytes during DN. Conditional Smad4 deletion in podocytes protects mice from type 2 DN, independent of obesity. Mechanistically, hyperglycaemia induces Smad4 localization to mitochondria in podocytes, resulting in reduced glycolysis and oxidative phosphorylation and increased production of reactive oxygen species. This operates, in part, via direct binding of Smad4 to the glycolytic enzyme PKM2 and reducing the active tetrameric form of PKM2. In addition, Smad4 interacts with ATPIF1, causing a reduction in ATPIF1 degradation. In conclusion, we have discovered a mitochondrial mechanism by which Smad4 causes diabetic podocyte injury., (© 2020 The Authors.)

Osteopontin (OPN) is an important component in both bone and blood regulation, functioning as a bridge between the two. Previously, thrombin-cleaved osteopontin (trOPN), the dominant form of OPN in adult bone marrow (BM), was demonstrated to be a critical negative regulator of adult hematopoietic stem cells (HSC) via interactions with α 4 β 1 and α 9 β 1 integrins. We now demonstrate OPN is also required for fetal hematopoiesis in maintaining the HSC and progenitor pool in fetal BM. Specifically, we showed that trOPN is highly expressed in fetal BM and its receptors, α 4 β 1 and α 9 β 1 integrins, are both highly expressed and endogenously activated on fetal BM HSC and progenitors. Notably, the endogenous activation of integrins expressed by HSC was attributed to high concentrations of three divalent metal cations, Ca 2+ , Mg 2+ and Mn 2+ , which were highly prevalent in developing fetal BM. In contrast, minimal levels of OPN were detected in fetal liver, and α 4 β 1 and α 9 β 1 integrins expressed by fetal liver HSC were not in the activated state, thereby permitting the massive expansion of HSC and progenitors required during early fetal hematopoiesis. Consistent with these results, no differences in the number or composition of hematopoietic cells in the liver of fetal OPN -/- mice were detected, but significant increases in the hematopoietic progenitor pool in fetal BM as well as an increase in the BM HSC pool following birth and into adulthood were observed. Together, the data demonstrates OPN is a necessary negative regulator of fetal and neonatal BM progenitors and HSC, and it exhibits preserved regulatory roles during early development, adulthood and ageing.

Acute kidney injury leading to chronic kidney disease through tubulointerstitial fibrosis is a major challenge in nephropathy. Several signaling pathways promote interstitial fibrosis; however, effective suppression of fibrosis may require blockade of more than one pathway. This study investigated whether blockade of Smad3 and c-Jun N-terminal kinase (JNK) signaling gives added suppression of interstitial fibrosis in folic acid nephropathy. A single high dose of folic acid (FA) causes acute tubular damage in C57BL/6J mice followed by interstitial fibrosis and chronic renal impairment. Co-activations of Smad3 and JNK signaling occur in both tubular epithelial cells and myofibroblasts in areas of tubulointerstitial damage and fibrosis in both murine FA-induced nephropathy and human IgA nephropathy. Groups of mice were treated with a Smad3 inhibitor (SIS3), a JNK inhibitor (SP600125), or a combination from day 6 after FA administration until being killed on day 28. Each drug efficiently inhibited its specific target (Smad3 phosphorylation or c-Jun phosphorylation) without affecting the other pathway. Given alone, each drug partially reduced renal fibrosis, whereas the combination therapy gave an additive and profound protection from renal fibrosis and improved renal function. Inhibition of Smad3 and/or JNK signaling activities prevented down-regulation of PGC-1α in tubular epithelial cells and up-regulation of PGC-1α in myofibroblasts during FA-induced renal fibrosis and inflammation. The expression of PGC-1α was upregulated in Smad3 -/- NRK52E cells while downregulated in Smad3 -/- NRK49F cells, suggesting that Smad3 signaling may regulate expression of PGC-1α in renal tubular epithelial cells and fibroblasts in distinct fashion. In vivo and cell culture studies also indicate that Smad3 and JNK signaling cooperate to cause mitochondrial dysfunction and cell damage in tubular epithelial cells via direct actions on the transcription of PGC-1α. These pathways also act cooperatively to promote renal fibroblast proliferation in tempo-spatial fashion. In conclusion, we have identified a potential combination therapy for progressive renal fibrosis which operates, in part, through modifying mitochondrial function.

The genetic background of a mouse strain determines its susceptibility to disease. C57BL/6J and Balb/CJ are two widely used inbred mouse strains that we found react dramatically differently to angiotensin II and high-salt diet (ANG II + Salt). Balb/CJ show increased mortality associated with anuria and edema formation while C57BL/6J develop arterial hypertension but do not decompensate and die. Clinical symptoms of heart failure in Balb/CJ mice gave the hypothesis that ANG II + Salt impairs cardiac function and induces cardiac remodeling in male Balb/CJ but not in male C57BL/6J mice. To test this hypothesis, we measured cardiac function using echocardiography before treatment and every day for 7 days during treatment with ANG II + Salt. Interestingly, pulsed wave Doppler of pulmonary artery flow indicated increased pulmonary vascular resistance and right ventricle systolic pressure in Balb/CJ mice, already 24 h after ANG II + Salt treatment was started. In addition, Balb/CJ mice showed abnormal diastolic filling indicated by reduced early and late filling and increased isovolumic relaxation time. Furthermore, Balb/CJ exhibited lower cardiac output compared with C57BL/6J even though they retained more sodium and water, as assessed using metabolic cages. Left posterior wall thickness increased during ANG II + Salt treatment but did not differ between the strains. In conclusion, ANG II + Salt treatment causes early restriction of pulmonary flow and reduced left ventricular filling and cardiac output in Balb/CJ, which results in fluid retention and peripheral edema. This makes Balb/CJ a potential model to study the adaptive capacity of the heart for identifying new disease mechanisms and drug targets.

Balb/CJ mice are more sensitive to treatment with angiotensin II (ANG II) and high-salt diet compared with C57BL/6J mice. Together with higher mortality, they develop edema, signs of heart failure, and acute kidney injury. The aim of the present study was to identify differences in renal gene regulation that may affect kidney function and fluid balance, which could contribute to decompensation in Balb/CJ mice after ANG II + salt treatment. Male Balb/CJ and C57BL/6J mice were divided into the following five different treatment groups: control, ANG II, salt, ANG II + salt, and ANG II + salt + N -acetylcysteine. Gene expression microarrays were used to explore differential gene expression after treatment and between the strains. Published data from the Mouse Genome Database were used to identify the associated genomic differences. The glomerular filtration rate (GFR) was measured using inulin clearance, and fluid balance was measured using metabolic cages. Gene ontology enrichment analysis of gene expression microarrays identified glutathione transferase (antioxidant system) as highly enriched among differentially expressed genes. Balb/CJ mice had similar GFR compared with C57BL/6J mice but excreted less Na + and water, although net fluid and electrolyte balance did not differ, suggesting that Balb/CJ mice may be inherently more prone to decompensation. Interestingly, C57BL/6J mice had higher urinary oxidative stress despite their relative protection from decompensation. In addition, treatment with the antioxidant N -acetylcysteine decreased oxidative stress in C57BL/6J mice, reduced urine excretion, and increased mortality. Balb/CJ mice are more sensitive than C57BL/6J to ANG II + salt, in part mediated by lower oxidative stress, which favors fluid and Na + retention.

Activity of lipoprotein lipase (LPL) is high in mouse kidney, but the reason is poorly understood. The aim was to characterize localization, regulation, and function of LPL in kidney of C57BL/6J mice. We found LPL mainly in proximal tubules, localized inside the tubular epithelial cells, under all conditions studied. In fed mice, some LPL colocalized with the endothelial markers CD31 and GPIHBP1 and could be removed by perfusion with heparin, indicating a vascular location. The role of angiopoietin-like protein 4 (ANGPTL4) for nutritional modulation of LPL activity was studied in wild-type and Angptl4 -/- mice. In Angptl4 -/- mice, kidney LPL activity remained high in fasted animals, indicating that ANGPTL4 is involved in suppression of LPL activity on fasting, like in adipose tissue. The amount of ANGPTL4 protein in kidney was low, and the protein appeared smaller in size, compared with ANGPTL4 in heart and adipose tissue. To study the influence of obesity, mice were challenged with high-fat diet for 22 wk, and LPL was studied after an overnight fast compared with fasted mice given food for 3 h. High-fat diet caused blunting of the normal adaptation of LPL activity to feeding/fasting in adipose tissue, but in kidneys this adaptation was lost only in male mice. LPL activity increases to high levels in mouse kidney after feeding, but as no difference in uptake of chylomicron triglycerides in kidneys is found between fasted and fed states, our data confirm that LPL appears to have a minor role for lipid uptake in this organ.

Background: Primary prevention of cardiovascular disease often fails because of poor adherence among practitioners and individuals to prevention guidelines. We aimed to investigate whether ultrasound-based pictorial information about subclinical carotid atherosclerosis, targeting both primary care physicians and individuals, improves prevention., Methods: Visualization of asymptomatic atherosclerotic disease for optimum cardiovascular prevention (VIPVIZA) is a pragmatic, open-label, randomised controlled trial that was integrated within the Västerbotten Intervention Programme, an ongoing population-based cardiovascular disease prevention programme in northern Sweden. Individuals aged 40, 50, or 60 years with one or more conventional risk factors were eligible to participate. Participants underwent clinical examination, blood sampling, and ultrasound assessment of carotid intima media wall thickness and plaque formation. Participants were randomly assigned 1:1 with a computer-generated randomisation list to an intervention group (pictorial representation of carotid ultrasound plus a nurse phone call to confirm understanding) or a control group (not informed). The primary outcomes, Framingham risk score (FRS) and European systematic coronary risk evaluation (SCORE), were assessed after 1 year among participants who were followed up. This study is registered with ClinicalTrials.gov, number NCT01849575., Findings: 3532 individuals were enrolled between April 29, 2013, and June 7, 2016, of which 1783 were randomly assigned to the control group and 1749 were assigned to the intervention group. 3175 participants completed the 1-year follow-up. At the 1-year follow-up, FRS and SCORE differed significantly between groups (FRS 1·07 [95% CI 0·11 to 2·03, p=0·0017] and SCORE 0·16 [0·02 to 0·30, p=0·0010]). FRS decreased from baseline to the 1-year follow-up in the intervention group and increased in the control group (-0·58 [95% CI -0·86 to -0·30] vs 0·35 [0·08 to 0·63]). SCORE increased in both groups (0·13 [95% CI 0·09 to 0·18] vs 0·27 [0·23 to 0·30])., Interpretation: This study provides evidence of the contributory role of pictorial presentation of silent atherosclerosis for prevention of cardiovascular disease. It supports further development of methods to reduce the major problem of low adherence to medication and lifestyle modification., Funding: Västerbotten County Council, the Swedish Research Council, the Heart and Lung Foundation, the Swedish Society of Medicine, and Carl Bennet Ltd, Sweden., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The tracking of the hematopoietic potential of genetically manipulated fluorescent hematopoietic stem cells (HSC) in the bone marrow (BM) allows the assessment of regulatory processes involved in the re-establishment of hematopoiesis posttransplant. Herein, we describe the means to assess the consequence of expressing specific genes in HSC on their engraftment potential posttransplant.

The coordination of the organ-specific responses regulating systemic energy distribution to replenish lipid stores in acutely activated brown adipose tissue (BAT) remains elusive. Here, we show that short-term cold exposure or acute β3-adrenergic receptor (β3AR) stimulation results in secretion of the anabolic hormone insulin. This process is diminished in adipocyte-specific Atgl -/- mice, indicating that lipolysis in white adipose tissue (WAT) promotes insulin secretion. Inhibition of pancreatic β cells abolished uptake of lipids delivered by triglyceride-rich lipoproteins into activated BAT. Both increased lipid uptake into BAT and whole-body energy expenditure in response to β3AR stimulation were blunted in mice treated with the insulin receptor antagonist S961 or lacking the insulin receptor in brown adipocytes. In conclusion, we introduce the concept that acute cold and β3AR stimulation trigger a systemic response involving WAT, β cells, and BAT, which is essential for insulin-dependent fuel uptake and adaptive thermogenesis., (Copyright © 2018 Elsevier Inc. All rights reserved.)