BACKGROUND & AIMS:The eastern woodchuck (Marmota monax), which can be naturally infected with woodchuck hepatitis virus (WHV), has served as a model for studying aspects of human HBV infection, including the establishment of chronic infection and progression from chronic hepatitis to liver cancer. However, the cellular landscape of the woodchuck liver and its parallels to HBV infection remain uncharacterized. METHODS:We generated a single-cell and spatial transcriptomic atlas of the woodchuck liver in health and chronic WHV infection, characterizing cell types and infection-associated processes in hepatic tissue (healthy: 52,024 cells; infected: 40,810 cells; n = 8 per group) and peripheral blood mononuclear cells (healthy: 25,314 cells, n = 7; infected: 19,518 cells, n = 8). We further examined shared WHV-HBV disease pathways transcriptionally and assessed woodchuck liver immune responses functionally using PCLS (precision-cut liver slice) stimulation. RESULTS:Using this atlas, we found that hepatic cellular and immune diversity in the woodchuck liver is comparable to that of human livers. Immune cells in PMA/ionomycin-stimulated PCLS exhibited a robust type I inflammatory response, as expected, supporting the accuracy of our cell-type annotations. Our atlas further revealed transcriptional and cellular similarities between WHV- and HBV-infected livers, including periportal dendritic cell activation and a restructuring of the T-cell compartment from memory toward exhaustion during WHV infection, a hallmark of chronic human HBV. CONCLUSIONS:We present a multi-omic atlas of healthy, diseased, and ex vivo-stimulated woodchuck liver. This work identified shared WHV-HBV pathological processes, reinforces the value of this preclinical model, and provides a resource to advance HBV pathogenesis studies and therapeutic development. IMPACT AND IMPLICATIONS:Limited treatment options for liver disease, often culminating in the need for liver transplantation, underscore the requirement for human-relevant animal models to speed up the development of new therapeutic interventions. Woodchucks infected with woodchuck hepatitis virus develop chronic hepatitis and liver cancer, mirroring human HBV infection. However, the cellular composition and active biological processes within the woodchuck liver have remained poorly understood, limiting the model's utility for therapeutic discovery. In this study, we characterize the healthy and chronically infected woodchuck liver at single-cell resolution and compare it with human HBV infection, thereby reinforcing the value of the WHV-infected woodchuck as a model for human HBV disease.

Endothelial function is a safeguard against atherosclerosis, yet endothelial‑to‑mesenchymal transition (EndMT) can occur at atheroprone sites. How endothelial identity loss contributes to atherogenesis remains unclear. Here we investigated the role of the ETS transcription factor ERG, a key regulator of endothelial identity, in atheroprogression. Inducible endothelial Erg deletion increased plaque burden in hypercholesterolemic mice. Lineage tracing and single‑cell transcriptomics showed that ERG loss drove endothelial dedifferentiation, mesenchymal fate acquisition, and migration and expansion of EndMT cells within plaques, which interacted with pro‑atherogenic cells. ERG loss also caused early disruption of barrier function, promoting lipid uptake and foam cell accumulation in normally atheroresistant regions. In human atherosclerotic plaques, ERG chromatin accessibility and expression were reduced, with transcriptional changes mirroring those in mice. Restoring ERG in cultured EndMT cells reversed mesenchymal programs and re‑established endothelial identity. These findings identify ERG as an enforcer of endothelial function that restricts EndMT to limit atherosclerosis.

Monocyte recruitment from the blood into tissues is a fundamental component of inflammatory conditions, including atherosclerosis. During recruitment, monocytes adherent to activated endothelium are exposed to external forces imparted by flowing blood. How they respond and resist detachment remains poorly understood. Using parallel plate flow chamber assays, we showed that force-dependent, α4 integrin-mediated monocyte spreading on VCAM-1 requires a Ca2+ influx that is mediated by the mechanosensitive ion channel Piezo1. In Ccr2-CreERT2; Piezo1fl/fl mice, conditional deletion of Piezo1 in Ly6Chi monocytes significantly lowered the number of monocytes and monocyte-derived macrophages in the peritoneal cavity following thioglycolate- or ovalbumin-induced peritonitis. Piezo1 deletion had no effect on the number of circulating blood monocytes. To study atherogenesis, we backcrossed Piezo1-floxed transgenics into the Ldlr–/– background. Deletion of monocyte Piezo1 in Ldlr–/– mice fed a cholesterol-rich diet reduced the size and macrophage content of 3-week atherosclerotic lesions. Neither the uptake of oxidized LDL nor phagocytosis were impaired in monocyte-derived Piezo1-deficient macrophages, suggesting that smaller lesions may be due to reduced monocyte recruitment primarily and not impaired macrophage lipid uptake. These findings provide novel molecular insights into the regulation of monocyte recruitment and demonstrate a role for monocyte Piezo1 in atherogenesis. Supported by CIHR FDN-154299 (M.I.C.). M.I.C. holds a Tier 1 Canada Research Chair. H.I. is supported by Queen Elizabeth II/ Heart and Stroke Foundation of Ontario Graduate Scholarship in Science and Technology. Cellular Adhesion, Migration, and Inflammation (CAM)

BACKGROUND & AIMS:Interleukin-4 (IL-4) is a key contributor to liver regeneration, but its effects remain poorly understood due to a lack of models that preserve the complex cellular interactions of the liver. Here, we use murine precision-cut liver slices (PCLS), a 3-dimensional tissue culture system that maintains both parenchymal and non-parenchymal cells, to investigate the role of IL-4 in hepatic cell reprogramming. Through longitudinal single-cell transcriptomics and protein-level validation, we demonstrate the proregenerative potential of IL-4. METHODS:We performed longitudinal single nucleus RNA sequencing on PCLS from 8- to 10-week-old C57BL/6 mice over 5 days of culture in the presence and absence of IL-4. We assessed intracellular ATP output to demonstrate slice viability. We further performed orthogonal evaluations of the impact of IL-4 treatment via immunhistochemical staining to confirm proliferation and cell identity within the slices. We then assessed the impact of IL-4 exposure in slices generated from the diseased livers (hepatonecrosis/fibrosis) of mice treated with thioacetamide. RESULTS:IL-4 induced transcriptional changes, including increased expression of tissue repair-associated markers in myeloid cells, expansion of hepatocyte and cholangiocyte progenitors, and inhibition of fibroblast activation. Additionally, IL-4 treatment significantly increased Ki67 protein expression and intracellular ATP production, indicating enhanced proliferation and viability. Notably, IL-4 also improved cellular viability in slices from thioacetamide-treated mice, highlighting its potential proregenerative effects in injured liver tissue. CONCLUSIONS:Our study highlights the potential of IL-4-driven modulation of the liver microenvironment, paving the way for cytokine-based therapeutic strategies to enhance immune-mediated hepatic regeneration.

Aim: The ETS transcription factor ERG has been identified as a principal regulator of endothelial function through its ability to repress inflammation in endothelial cells (ECs), and loss of ERG induces endothelial to mesenchymal transition (EndMT) in fibrotic disease. To investigate ERG function in atherosclerosis, we employed an EC-specific Erg knockout ( Erg EC - KO ) in PCSK9-overexpression and apoE-deficient murine atherosclerosis models. Methods: Atherosclerosis was induced in 10-week-old Erg EC - KO mice or wild-type counterparts that had a Cre-inducible EC lineage tag ( Cdh5 CreER ; R26-CAG-LSL-Sun1-sfGFP ). Atherosclerosis was modeled by Apoe deletion or AAV8-PCSK9 with 12 weeks of high-cholesterol diet (1.25%). Aortic intimal cells were assessed with flow cytometry. Plaque burden, morphology, and cellularity were characterized with brightfield, Oil Red O, H&E, Movat, and immunofluorescence imaging. Results: Erg EC-KO in PCSK9-overexpression mice led to a 1.6-fold increase in aortic plaque burden and plaque formation in regions that are usually protected (e.g., greater curvature; p≤0.03; n=9-10). Loss of ERG increased elastin breaks (1.7-fold; p=0.02; n=9) and decreased fibrous cap thickness (0.76-fold; p=0.02; n=9-10) in aortic arch cross sections. Aortic intimal digests from Erg EC- KO mice contained increased Sun1-sfGFP + EC lineage cells (1.9-fold; p=0.01; n=4), which had reduced CD31 positivity (0.79-fold; p=0.03; n=4). Notably, a 5.3-fold increase in EC lineage cells was observed in plaques from brachiocephalic artery cross sections (p=0.0001; n=6-15), which had hallmarks of EndMT (e.g., 31.5-fold increase in GFP + ACTA2 + cells, p=0.02, n=3-5). Increased aortic plaque burden was recapitulated in apoE-deficient E rg EC- KO mice (2.1-fold; p=0.02; n=4-8). Conclusion: Endothelial Erg KO results in increased plaque burden and altered plaque topography in murine atherosclerosis. Loss of ERG induces EndMT and marked ingression of ECs from the luminal endothelium to the plaque interior. These findings highlight loss of ERG as a novel mechanism by which intimal ECs acquire mesenchymal identity in atherosclerosis and implicate EC identity loss as a driver of EC migration into plaques, which is associated with a vulnerable morphology.

BACKGROUND:Myeloid cells (MCs) reside in the aortic intima at regions predisposed to atherosclerosis. Systemic inflammation triggers reverse transendothelial migration (RTM) of intimal MCs into the arterial blood, which orchestrates a protective immune response that clears intracellular pathogens from the arterial intima. Molecular pathways that regulate RTM remain poorly understood. S1P (sphingosine-1-phosphate) is a lipid mediator that regulates immune cell trafficking by signaling via 5 G-protein-coupled receptors (S1PRs [S1P receptors]). We investigated the role of S1P in the RTM of aortic intimal MCs.METHODS:Intravenous injection of lipopolysaccharide was used to model a systemic inflammatory stimulus that triggers RTM. CD11c+ intimal MCs in the lesser curvature of the ascending aortic arch were enumerated by en face confocal microscopy. Local gene expression was evaluated by transcriptomic analysis of microdissected intimal cells.RESULTS:In wild-type C57BL/6 mice, lipopolysaccharide induced intimal cell expression of S1pr1, S1pr3, and Sphk1 (a kinase responsible for S1P production). Pharmacological modulation of multiple S1PRs blocked lipopolysaccharide-induced RTM and modulation of S1PR1 and S1PR3 reduced RTM in an additive manner. Cre-mediated deletion of S1pr1 in MCs blocked lipopolysaccharide-induced RTM, confirming a role for myeloid-specific S1PR1 signaling. Global or hematopoietic deficiency of Sphk1 reduced plasma S1P levels, the abundance of CD11c+ MCs in the aortic intima, and blunted lipopolysaccharide-induced RTM. In contrast, plasma S1P levels, the abundance of intimal MCs, and lipopolysaccharide-induced RTM were rescued in Sphk1-/- mice transplanted with Sphk1+/+ or mixed Sphk1+/+ and Sphk1-/- bone marrow. Stimulation with lipopolysaccharide increased endothelial permeability and intimal MC exposure to circulating factors such as S1P.CONCLUSIONS:Functional and expression studies support a novel role for S1P signaling in the regulation of lipopolysaccharide-induced RTM and the homeostatic maintenance of aortic intimal MCs. Our data provide insight into how circulating plasma mediators help orchestrate intimal MC dynamics.

The accumulation of lipid and the formation of macrophage foam cells is a hallmark of atherosclerosis, a chronic inflammatory disease. To better understand the role of macrophage lipid accumulation in inflammation during atherogenesis, we studied early molecular events that follow the accumulation of oxidized low-density lipoprotein (oxLDL) in cultured mouse macrophages. We previously showed that oxLDL accumulation downregulates the inflammatory response in conjunction with downregulation of late-phase glycolysis. In this study, we show that within hours after LPS stimulation, macrophages with accumulated oxLDL maintain early-phase glycolysis but selectively downregulate activation of AKT2, one of three AKT isoforms. The inhibition of AKT2 activation reduced LPS-induced ATP citrate lyase activation, acetyl-CoA production, and acetylation of histone 3 lysine 27 (H3K27ac) in certain inflammatory gene promoters. In contrast to oxLDL, multiple early LPS-induced signaling pathways were inhibited in macrophages with accumulated cholesterol, including TBK1, AKT1, AKT2, MAPK, and NF-κB, and early-phase glycolysis. The selective inhibition of LPS-induced AKT2 activation was dependent on the generation of mitochondrial oxygen radicals during the accumulation of oxLDL in macrophages prior to LPS stimulation. This is consistent with increased oxidative phosphorylation, fatty acid synthesis, and oxidation pathways found by comparative transcriptomic analyses of oxLDL-loaded versus control macrophages. Our study shows a functional connection between oxLDL accumulation, inactivation of AKT2, and the inhibition of certain inflammatory genes through epigenetic changes that occur soon after LPS stimulation, independent of early-phase glycolysis.

High-resolution single-cell technologies have shed light on the pathogenesis of cardiovascular diseases by enabling the discovery of novel cellular and transcriptomic signatures associated with various conditions, and uncovering new contributions of inflammatory processes, immunity, metabolic stress, and risk factors. We review the information obtained from studies using single-cell technologies in tissues with atherosclerosis and aortic aneurysms. Insights are provided on the biology of endothelial, smooth muscle, and immune cells in the arterial intima and media. In addition to cellular diversity, numerous examples of plasticity and phenotype switching are highlighted and presented in the context of normal cell functions.

Hypercholesterolemia (HC) is a key risk factor for atherosclerosis, a chronic inflammatory disease that causes myocardial infarction and stroke. Although all regions of arteries are exposed to HC, atherosclerosis develops in discrete regions, such as the lesser curvature (LC) of the mouse aortic arch. In Ldlr -/- mice HC induces intimal myeloid cells to develop into the foam cells of nascent lesions. The purpose of this study is to characterize the transcriptomic changes occurring in the aortic intimal myeloid cells during the earliest stages of atherogenesis, and correlate them to cellular responses. Intimal cells were isolated from the LC of the ascending aortic arch of Ldlr -/- mice at 0, 5, 14, and 56 days of CRD feeding using en face enzymatic digestion and cell microisolation. Bulk RNA-seq and RT-qPCR revealed that genes associated with lipid-loaded macrophages (e.g., Abcg1 , Lgals3 ) were progressively elevated over time. Atherogenesis-associated inflammatory transcripts and pathways, such as leukocyte transendothelial migration, cell adhesion, and cytokine/chemokine signalling were significantly elevated after 14d of CRD, but not at 5d. Single cell transcriptomic analysis of the mouse aortic arch intima of Ldlr -/- mice revealed multiple sub-populations of myeloid cells. With our intima specific approach, we have uncovered a diverse landscape of intimal myeloid cells; including monocytes, macrophages, foam cells, DCs, and neutrophils. Atherogenesis-associated inflammatory transcripts were scarcely detected in macrophage and foam cell clusters, but were prominent in non-macrophage cell types. Ccr2 + intimal monocytes exhibited a hypercholesterolemia induced increase in inflammatory pathways within 5 days of CRD feeding. Furthermore, analysis of monocyte and macrophage clusters suggests that this inflammatory signature dissipates as recently recruited monocytes transition into a macrophage/foam cell fate. We are using Ccr2 + fate mapping models, to interrogate the hypercholesterolemia-induced transition of inflammatory monocytes within a growing lesion. We suggest that the atherogenesis-associated inflammation may be initiated by recently recruited monocytes, and may not originate directly from lipid loaded macrophages.

Lipid accumulation in macrophages (Mφs) is a hallmark of atherosclerosis, yet how lipid accumulation affects inflammatory responses through rewiring of Mφ metabolism is poorly understood. We modeled lipid accumulation in cultured wild-type mouse thioglycolate-elicited peritoneal Mφs and bone marrow-derived Mφs with conditional (Lyz2-Cre) or complete genetic deficiency of Vhl, Hif1a, Nos2, and Nfe2l2. Transfection studies employed RAW264.7 cells. Mφs were cultured for 24 h with oxidized low-density lipoprotein (oxLDL) or cholesterol and then were stimulated with LPS. Transcriptomics revealed that oxLDL accumulation in Mφs downregulated inflammatory, hypoxia, and cholesterol metabolism pathways, whereas the antioxidant pathway, fatty acid oxidation, and ABC family proteins were upregulated. Metabolomics and extracellular metabolic flux assays showed that oxLDL accumulation suppressed LPS-induced glycolysis. Intracellular lipid accumulation in Mφs impaired LPS-induced inflammation by reducing both hypoxia-inducible factor 1-α (HIF-1α) stability and transactivation capacity; thus, the phenotype was not rescued in Vhl-/- Mφs. Intracellular lipid accumulation in Mφs also enhanced LPS-induced NF erythroid 2-related factor 2 (Nrf2)-mediated antioxidative defense that destabilizes HIF-1α, and Nrf2-deficient Mφs resisted the inhibitory effects of lipid accumulation on glycolysis and inflammatory gene expression. Furthermore, oxLDL shifted NADPH consumption from HIF-1α- to Nrf2-regulated apoenzymes. Thus, we postulate that repurposing NADPH consumption from HIF-1α to Nrf2 transcriptional pathways is critical in modulating inflammatory responses in Mφs with accumulated intracellular lipid. The relevance of our in vitro models was established by comparative transcriptomic analyses, which revealed that Mφs cultured with oxLDL and stimulated with LPS shared similar inflammatory and metabolic profiles with foamy Mφs derived from the atherosclerotic mouse and human aorta.

Macrophage colony stimulating factor-1 (CSF-1) plays a critical role in maintaining myeloid lineage cells. However, congenital global deficiency of CSF-1 (Csf1op/op) causes severe musculoskeletal defects that may indirectly affect hematopoiesis. Indeed, we show here that osteolineage-derived Csf1 prevented developmental abnormalities but had no effect on monopoiesis in adulthood. However, ubiquitous deletion of Csf1 conditionally in adulthood decreased monocyte survival, differentiation, and migration, independent of its effects on bone development. Bone histology revealed that monocytes reside near sinusoidal endothelial cells (ECs) and leptin receptor (Lepr)-expressing perivascular mesenchymal stromal cells (MSCs). Targeted deletion of Csf1 from sinusoidal ECs selectively reduced Ly6C- monocytes, whereas combined depletion of Csf1 from ECs and MSCs further decreased Ly6Chi cells. Moreover, EC-derived CSF-1 facilitated recovery of Ly6C- monocytes and protected mice from weight loss following induction of polymicrobial sepsis. Thus, monocytes are supported by distinct cellular sources of CSF-1 within a perivascular BM niche.

Hypercholesterolemia (HC) is a key risk factor of atherosclerosis, a chronic inflammatory disease that causes myocardial infarction and stroke. Although all regions of arteries are exposed to HC, atherosclerosis develops in discrete regions, such as the lesser curvature (LC) of the mouse aortic arch. Dietary HC in Ldlr -/- mice induces intimal myeloid cells to develop into foam cells, constituting nascent lesions. The purpose of this study is to characterize the transcriptomic changes occurring in the aortic intima during the earliest stages of atherogenesis, and correlate them to cellular responses. Using en face enzymatic digestion and intimal cell microisolation, transcriptomic analysis was performed on the LC. Intimal cells were isolated from the LC of the ascending aortic arch of Ldlr -/- mice at 0, 5, and 14 days of CRD feeding. Bulk RNA-seq and RT-qPCR found that genes associated with lipid-loaded macrophages (e.g., Abcg1, Lgals3 ) were progressively elevated at 5 and 14 days of CRD. Atherogenesis-associated inflammatory transcripts and pathways, such as leukocyte transendothelial migration, cell adhesion, and cytokine/chemokine signalling were significantly elevated after 14d of CRD, but not detected at 5d. Single cell transcriptomic analysis of the mouse aortic arch intima of Ldlr -/- mice revealed multiple sub-populations of myeloid, endothelial, and other cell types. A shift in the C1qb + , Mmp12 + myeloid subpopulations was observed as the duration of cholesterol feeding increased - with expansion of a macrophage population rich in lipid-responsive transcripts. This finding is consistent with an increase in macrophage number and the presence of Nile Red + CD68 + foam cells observed by immunoconfocal microscopy at 5 days of CRD. However, a robust inflammatory signature was not detected in this macrophage cluster. Interestingly, an inflammatory signature similar to that reported in early atherogenesis was identified in a distinct Ccr2 + myeloid population with no signature of lipid loading. This suggests that the inflammation associated with atherogenesis may be a result of intercellular crosstalk between distinct myeloid subpopulations within the intima and may not originate directly from lipid loaded macrophages.

CD11c + myeloid cells (MCs) reside in the normal aortic arch intima at regions predisposed to atherosclerosis. We showed that reverse transendothelial migration (RTM) of these intimal MCs into the arterial circulation is a protective immune response. RTM is triggered by systemic stimulation of cytokine and toll-like receptors (TLRs), e.g., TLR4 by lipopolysaccharide (LPS), and is inhibited by hypercholesterolemia and associated intimal MC lipid accumulation. We hypothesize that signaling by sphingosine-1-phosphate (S1P) and its receptors (S1PRs) is required for RTM of intimal MCs following systemic stimulation of TLRs, analogous to its role in immune cell trafficking in lymphoid tissues. In the lesser curvature of the aortic arch of wild type C57BL/6 mice, LPS induced the expression of S1P receptors 1 & 3, and sphingosine kinase 1 (SphK1). FTY720, a broad-spectrum S1P receptor modulator, blocked LPS-induced RTM, as determined by the enumeration of CD11c+ MCs by en face immunoconfocal microscopy. S1PR1 or S1PR3 inhibitors blocked RTM in a partial and additive manner, suggesting a role for both S1P receptors in intimal MC migration. Initial experiments show that Cre-mediated deletion of S1PR1 in myeloid cells partially blocks LPS-induced RTM, suggesting that myeloid-specific S1PR1 signaling is implicated in the migratory process. Furthermore, global loss or hematopoietic deficiency of SPHK1 reduced the abundance of CD11c+ MCs in the aortic intima, suggesting an additional role for myeloid S1P production in the homeostatic maintenance of resident aortic immune cells. Cre-mediated deletion of S1PR1 in endothelial cells also reduced the abundance of intimal MCs, providing further evidence that S1P signaling in the intima is important in maintaining the intimal MC population. Thus, functional and expression studies suggest that S1P signaling plays a role in both the maintenance of resident intimal MCs and LPS-induced RTM.

One of the hallmarks of atherosclerosis is ongoing accumulation of macrophages in the artery intima beginning at disease onset. Monocyte recruitment contributes to increasing macrophage abundance at early stages of atherosclerosis. Although the chemokine CCL5 (RANTES) has been studied in atherosclerosis, its role in the recruitment of monocytes to early lesions has not been elucidated. We show that expression of Ccl5 mRNA, as well as other ligands of the CCR5 receptor (Ccl3 and Ccl4), is induced in the aortic intima of Ldlr-/- mice 3 weeks after the initiation of cholesterol-rich diet (CRD)-induced hypercholesterolemia. En face immunostaining revealed that CCL5 protein expression is also upregulated at 3 weeks of CRD. Blockade of CCR5 significantly reduced monocyte recruitment to 3-week lesions, suggesting that chemokine signaling through CCR5 is critical. However, we observed that Ccl5-deficiency had no effect on early lesion formation and CCL5-blockade did not affect monocyte recruitment in Ldlr-/- mice. Immunostaining of the lesions in Ldlr-/- mice and reciprocal bone marrow transplantation (BMT) of Ccl5+/+ and Ccl5-/- mice revealed that CCL5 is expressed by both myeloid and endothelial cells. BMT experiments were carried out to determine if CCL5 produced by distinct cells has functions that may be concealed in Ccl5- /-Ldlr-/- mice. We found that hematopoietic cell-derived CCL5 regulates monocyte recruitment and the abundance of intimal macrophages in 3-week lesions of Ldlr- /- mice but plays a minor role in 6-week lesions. Our findings suggest that there is a short window in early lesion formation during which myeloid cell-derived CCL5 has a critical role in monocyte recruitment and macrophage abundance.

Abstract Background Sensitive, rapid, and accessible diagnostics continue to be critical to track the COVID-19 pandemic caused by the SARS-CoV-2 virus. RT-qPCR is the gold standard test, and comparison of methodologies and reagents, utilizing patient samples, is important to establish reliable diagnostic pipelines. Methods Here, we assessed indirect methods that require RNA extraction with direct RT-qPCR on patient samples. Four different RNA extraction kits (Qiagen, Invitrogen, BGI and Norgen Biotek) were compared. For detection, we assessed two recently developed Taqman-based modules (BGI and Norgen Biotek), a SYBR green-based approach (NEB Luna Universal One-Step Kit) with published and newly-developed primers, and clinical results (Seegene STARMag RNA extraction system and Allplex 2019-nCoV RT-qPCR assay). We also tested and optimized direct, extraction-free detection using these RT-qPCR systems and performed a cost analysis of the different methods evaluated here. Results Most RNA isolation procedures performed similarly, and while all RT-qPCR modules effectively detected purified viral RNA, the BGI system provided overall superior performance (lower detection limit, lower Ct values and higher sensitivity), generating comparable results to original clinical diagnostic data, and identifying samples ranging from 65 copies to 2.1 × 105 copies of viral genome/μl. However, the BGI detection system is more expensive than other options tested here. With direct RT-qPCR, simply adding an RNase inhibitor greatly improved detection, without the need for any other treatments (e.g. lysis buffers or boiling). The best direct methods detected ~ 10 fold less virus than indirect methods, but this simplified approach reduced sample handling, as well as assay time and cost. Conclusions With extracted RNA, the BGI RT-qPCR detection system exhibited superior performance over the Norgen system, matching initial clinical diagnosis with the Seegene Allplex assay. The BGI system was also suitable for direct, extraction-free analysis, providing 78.4% sensitivity. The Norgen system, however, still accurately detected samples with a clinical Ct < 33 from extracted RNA, provided significant cost savings, and was superior to SYBR green assays that exhibited reduced specificity.

Rationale: Bone marrow transplantation (BMT) is used frequently to study the role of hematopoietic cells in atherosclerosis, but aortic arch lesions are smaller in mice after BMT. Objective: To identify the earliest stage of atherosclerosis inhibited by BMT and elucidate potential mechanisms. Methods and Results: Ldlr −/− mice underwent total body γ-irradiation, bone marrow reconstitution, and 6-week recovery. Atherosclerosis was studied in the ascending aortic arch and compared with mice without BMT. In BMT mice, neutral lipid and myeloid cell topography were lower in lesions after feeding a cholesterol-rich diet for 3, 6, and 12 weeks. Lesion coalescence and height were suppressed dramatically in mice post-BMT, whereas lateral growth was inhibited minimally. Targeted radiation to the upper thorax alone reproduced the BMT phenotype. Classical monocyte recruitment, intimal myeloid cell proliferation, and apoptosis did not account for the post-BMT phenotype. Neutral lipid accumulation was reduced in 5-day lesions, thus we developed quantitative assays for LDL (low-density lipoprotein) accumulation and paracellular leakage using DiI-labeled human LDL and rhodamine B-labeled 70 kD dextran. LDL accumulation was dramatically higher in the intima of Ldlr −/− relative to Ldlr +/+ mice, and was inhibited by injection of HDL mimics, suggesting a regulated process. LDL, but not dextran, accumulation was lower in mice post-BMT both at baseline and in 5-day lesions. Since the transcript abundance of molecules implicated in LDL transcytosis was not significantly different in the post-BMT intima, transcriptomics from whole aortic arch intima, and at single-cell resolution, was performed to give insights into pathways modulated by BMT. Conclusions: Radiation exposure inhibits LDL entry into the aortic intima at baseline and the earliest stages of atherosclerosis. Single-cell transcriptomic analysis suggests that LDL uptake by endothelial cells is diverted to lysosomal degradation and reverse cholesterol transport pathways. This reduces intimal accumulation of lipid and impacts lesion initiation and growth.

The COVID-19 pandemic caused by the SARS-CoV-2 virus has placed extensive strain on RNA isolation and RT-qPCR reagents. Rapid development of new test kits has helped to alleviate these shortages. However, comparisons of these new detection systems are largely lacking. Here, we compare indirect methods that require RNA extraction, and direct RT-qPCR on patient samples. For RNA isolation we compared four different companies (Qiagen, Invitrogen, BGI and Norgen Biotek). For detection we compared two recently developed Taqman-based modules (BGI and Norgen Biotek), a SYBR green-based approach (NEB Luna Universal One-Step Kit) with published and newly-developed primers, and clinical results (Seegene STARMag RNA extraction system and Allplex 2019-nCoV RT-qPCR assay). Most RNA isolation procedures performed similarly, and while all RT-qPCR modules effectively detected purified viral RNA, the BGI system proved most sensitive, generating comparable results to clinical diagnostic data, and identifying samples ranging from 65 copies – 2.1×105 copies of viral Orf1ab/μl. However, the BGI detection system is ∼4x more expensive than other options tested here. With direct RT-qPCR we found that simply adding RNase inhibitor greatly improved sensitivity, without need for any other treatments (e.g. lysis buffers or boiling). The best direct methods were ∼10 fold less sensitive than indirect methods, but reduce sample handling, as well as assay time and cost. These studies will help guide the selection of COVID-19 detection systems and provide a framework for the comparison of additional systems.

Background/Rationale: Hypercholesterolemia (HC) is a key risk factor of atherosclerosis, a chronic inflammatory disease that causes myocardial infarction and stroke. Although all regions of arterie...

The bone marrow niche factors that sustain monocytes – phagocytes with complex functions in the circulation and peripheral tissues – and the functionally important cells that produce them, are poorly defined. Here, we conditionally deleted macrophage colony stimulating factor 1 (Csf1) in adult mice and show that CSF1 regulated differentiation and survival of monocytes and their precursor cells independent of its effects on bone development. Specifically, CSF1 produced by sinusoidal endothelial cells (ECs) but not leukocytes or stromal cells selectively maintained abundance of nonclassical Ly6Cnegative monocytes. Ly6Chigh monocytes, on the other hand, required CSF1 produced by ECs as well as leptin receptor (Lepr)-expressing perivascular stromal cells. Importantly, osteoblast-derived CSF1 critically supported bone formation and hematopoiesis during early development but did not contribute to maintenance of monocytes in adulthood. Our findings reveal classical and non-classical monocytes receive support by distinct cellular sources of CSF1 within a perivascular bone marrow niche.