High-mobility group box 1 fragment ameliorates chronic pancreatitis induced by caerulein in mice.

BACKGROUND: Chronic pancreatitis (CP) is a progressive disease characterized by pancreatic fibrosis for which effective treatment options are lacking. Mesenchymal stem cells (MSCs) have shown potential for fibrosis treatment but face limitations in clinical application. The high-mobility group box 1 (HMGB1) fragment mobilizes MSCs from bone marrow into the blood and has emerged as a promising therapeutic agent for tissue regeneration in various pathological conditions. The aim of this study was to investigate the potential therapeutic effects of systemic administration of the HMGB1 fragment in a mouse model of CP. METHODS: A caerulein-induced CP mouse model was used, and the HMGB1 fragment was administered by tail vein injection. Parameters such as body weight, pancreatic tissue damage, fibrosis, inflammatory cytokine expression, and collagen-related gene expression were evaluated using various assays, including immunohistochemistry, real-time PCR, serum analysis, and single-cell transcriptome analysis. And the migration of MSCs to the pancreas was evaluated using the parabiosis model. RESULTS: Administration of the HMGB1 fragment was associated with significant improvements in pancreatic tissue damage and fibrosis. It suppressed the expression of inflammatory cytokines and activated platelet-derived growth factor receptor-α+ MSCs, leading to their accumulation in the pancreas. The HMGB1 fragment also shifted gene expression patterns associated with pancreatic fibrosis toward those of the normal pancreas. Systemic administration of the HMGB1 fragment demonstrated therapeutic efficacy in attenuating pancreatic tissue damage and fibrosis in a CP mouse model. CONCLUSION: These findings highlight the potential of the HMGB1 fragment as a therapeutic target for the treatment of CP.

PDGFRα-lineage origin directs monocytes to trafficking proficiency to support peripheral immunity.

Multiple embryonic precursors give rise to leukocytes in adults while the lineage-based functional impacts are underappreciated. Mesodermal precursors expressing PDGFRα appear transiently during E7.5-8.5 descend to a subset of Lin- Sca1+ Kit+ hematopoietic progenitors found in adult BM. By analyzing a PDGFRα-lineage tracing mouse line, we here report that PDGFRα-lineage BM F4/80+ SSClo monocytes/macrophages are solely Ly6C+ LFA-1hi Mac-1hi monocytes enriched on the abluminal sinusoidal endothelium while Ly6C- LFA-1lo Mac-1lo macrophages are mostly from non-PDGFRα-lineage in vivo. Monocytes with stronger integrin profiles outcompete macrophages for adhesion on an endothelial monolayer or surfaces coated with ICAM-1-Fc or VCAM-1-Fc. Egress of PDGFRα-lineage-rich monocytes and subsequent differentiation to peripheral macrophages spatially segregates them from non-PDGFRα-lineage BM-resident macrophages and allows functional specialization since macrophages derived from these egressing monocytes differ in morphology, phenotype, and functionality from BM-resident macrophages in culture. Extravasation preference for blood PDGFRα-lineage monocytes varies by tissues and governs the local lineage composition of macrophages. More PDGFRα-lineage classical monocytes infiltrated into skin and colon but not into peritoneum. Accordingly, transcriptomic analytics indicated augmented inflammatory cascades in dermatitis skin of BM-chimeric mice harbouring only PDGFRα-lineage leukocytes. Thus, the PDGFRα-lineage origin biasedly generates monocytes predestined for BM exit to support peripheral immunity following extravasation and macrophage differentiation.

Longitudinal Single-Cell Transcriptomics Reveals a Role for Serpina3n-Mediated Resolution of Inflammation in a Mouse Colitis Model.

BACKGROUND & AIMS: Proper resolution of inflammation is essential to maintaining homeostasis, which is important as a dysregulated inflammatory response has adverse consequences, even being regarded as a hallmark of cancer. However, our picture of dynamic changes during inflammation remains far from comprehensive. METHODS: Here we used single-cell transcriptomics to elucidate changes in distinct cell types and their interactions in a mouse model of chemically induced colitis. RESULTS: Our analysis highlights the stromal cell population of the colon functions as a hub with dynamically changing roles over time. Importantly, we found that Serpina3n, a serine protease inhibitor, is specifically expressed in stromal cell clusters as inflammation resolves, interacting with a potential target, elastase. Indeed, genetic ablation of the Serpina3n gene delays resolution of induced inflammation. Furthermore, systemic Serpina3n administration promoted the resolution of inflammation, ameliorating colitis symptoms. CONCLUSIONS: This study provides a comprehensive, single-cell understanding of cell-cell interactions during colorectal inflammation and reveals a potential therapeutic target that leverages inflammation resolution.

Contribution of PDGFRα lineage cells in adult mouse hematopoiesis.

Platelet-derived growth factor receptor alpha (PDGFRα) is a dominant marker of mesodermal mesenchymal cells in mice. Previous studies demonstrated that PDGFRα-positive (PDGFRα+) mesodermal cells develop not only into mesenchymal cells but also into a subset of total hematopoietic cells (HCs) in the limited period during mouse embryogenesis. However, the precise characteristics of the PDGFRα lineage positive (PDGFRα Lin+) HCs in adult mouse hematopoiesis are largely unknown. In this study, we systematically evaluated the characteristics of PDGFRα Lin+ HCs in the bone marrow and peripheral blood using PDGFRα-CRE; ROSAtdTomato mice. Flow cytometry analysis revealed that PDGFRα Lin+ HCs accounted for approximately 20% of total HCs in both the bone marrow and peripheral blood in adult mice. Compositions of myeloid and lymphoid subpopulations among CD45+ mononuclear cells were almost identical in both PDGFRα Lin+ and PDGFRα Lin- cells. Single-cell RNA-sequencing analysis also demonstrated that the transcriptomic signatures of the PDGFRα Lin+ HCs in the peripheral blood largely overlapped with those of the PDGFRα Lin- HCs, suggesting equivalent functions of the PDGFRα Lin+ and PDGFRα Lin- HCs. Although pathophysiological activities of the PDGFRα Lin + HCs were not evaluated, our data clearly demonstrate a significant role of the PDGFRα Lin + HCs in physiological hematopoiesis in adult mice.

Lipid peroxidation increases hydrogen peroxide permeability leading to cell death in cancer cell lines that lack mtDNA.

4-Hydroxynonenal (HNE) is an important product of plasma membrane lipid peroxidation, which is a cause of cell and tissue injury. Mitochondrial DNA (mtDNA)-depleted ρ0 cells were established using human cervical cancer and oral squamous cell carcinoma cell lines. We investigated the effect of reactive oxygen species in ρ0 cells, especially the mechanism of hydrogen peroxide (H2 O2 )-mediated cell death. These cell were subjected to high oxidative stress and, compared with their parental cells, showed greater sensitivity to H2 O2 and high lipid peroxidation. Upregulation of HNE in the plasma membrane was observed prior to the increase in intracellular H2 O2 . The amount of oxidized lipid present changed H2 O2 permeability and administration of oxidized lipid led to further cell death after treatment with H2 O2 . Expression levels of lipoxygenase ALOX genes (ie ALOX5, ALOX12, and ALOX15) were upregulated in ρ0 cells, as were expression levels of ALOX12 and ALOX15 proteins. ALOX5 protein was mainly distributed in the nucleus, while ALOX12 and ALOX15 proteins were distributed in the nucleus and the cytoplasm. Although expression of COX2 gene was upregulated, its protein expression did not increase. ALOX (especially ALOX15) may be involved in the sensitivity of cancer cells to treatment. These data offer promise for the development of novel anticancer agents by altering the oxidation state of the plasma membrane. Our results showed that lipid peroxidation status is important for H2 O2 sensitivity and that ALOX15 is involved in lipid peroxidation status.

Transcriptionally distinct mesenchymal stem/stromal cells circulate in fetus.

Umbilical cord blood contains mesenchymal stem/stromal cells (MSCs) in addition to hematopoietic stem cells, serving as an attractive tool for regenerative medicine. As umbilical cord blood originates from fetus, abundant MSCs are expected to circulate in fetus. However, the properties of circulating MSCs in fetus have not been fully examined. In the present study, we aimed to analyze circulating MSCs, marked by the expression of platelet-derived growth factor receptor α (PDGFRα), during fetal development. Using PDGFRα GFP knock-in mice, we quantified the number of circulating PDGFRα positive MSCs during development. We further performed whole transcriptome analysis of circulating MSCs at single cell levels. We found that abundant PDGFRα positive cells circulate in embryo and diminish immediately after birth. In addition, single cell RNA-sequencing revealed transcriptional heterogeneity of MSCs in fetal circulation. These data lay a foundation to analyze the function of circulating MSCs during development.

Chromatin accessibility identifies diversity in mesenchymal stem cells from different tissue origins.

Mesenchymal stem cells (MSCs), which can differentiate into tri-lineage (osteoblast, adipocyte, and chondrocyte) and suppress inflammation, are promising tools for regenerative medicine. MSCs are phenotypically diverse based on their tissue origins. However, the mechanisms underlying cell-type-specific gene expression patterns are not fully understood due to the lack of suitable strategy to identify the diversity. In this study, we investigated gene expression programs and chromatin accessibilities of MSCs by whole-transcriptome RNA-seq analysis and an assay for transposase-accessible chromatin using sequencing (ATAC-seq). We isolated MSCs from four tissues (femoral and vertebral bone marrow, adipose tissue, and lung) and analysed their molecular signatures. RNA-seq identified the expression of MSC markers and both RNA-seq and ATAC-seq successfully clustered the MSCs based on their tissue origins. Interestingly, clustering based on tissue origin was more accurate with chromatin accessibility signatures than with transcriptome profiles. Furthermore, we identified transcription factors potentially involved in establishing cell-type specific chromatin structures. Thus, epigenome analysis is useful to analyse MSC identity and can be utilized to characterize these cells for clinical use.

Novel missense mutation in DLL4 in a Japanese sporadic case of Adams-Oliver syndrome.

Adams-Oliver syndrome (AOS, OMIM; 100300) is a rare genetic disease characterized by aplasia cutis congenita, terminal transverse limb defects and cutis marmorata with vascular anomalies such as congenital heart defects. The etiology of this syndrome has remained largely unknown but defective Notch signaling during vascular formation has been suggested. Here we describe a sporadic Japanese newborn case with clinically diagnosed AOS. Trio whole-exome sequencing identified a de novo, novel, heterozygous missense mutation in the Delta-like 4 ligand gene (DLL4 c.572G>A, p.Arg191His) in the patient. DLL4 functions as a requisite ligand for NOTCH1 receptor, which is essential for vascular formation. Amino acid substitution of Arg191 to His was predicted by molecular models to interfere with direct binding between DLL4 and NOTCH1. DLL4 has recently been identified as a causative gene of an autosomal dominant type of AOS with milder symptoms. The case described here showed gradual recovery from skull defects after birth and no psychomotor developmental delay has been observed. This is the second report of an AOS case with DLL4 mutation, and the phenotypic characteristics between the two cases are compared and discussed.

Quantitative determination of polysulfide in albumins, plasma proteins and biological fluid samples using a novel combined assays approach.

Hydrogen sulfide (H2S) signaling involves polysulfide (RSSnSR') formation on various proteins. However, the current lack of sensitive polysulfide detection assays poses methodological challenges for understanding sulfane sulfur homeostasis and signaling. We developed a novel combined assay by modifying Sulfide Antioxidant Buffer (SAOB) to produce an "Elimination Method of Sulfide from Polysulfide" (EMSP) treatment solution that liberates sulfide, followed with methylene blue (MB) sulfide detection assay. The combined EMSP-MB sulfide detection assay performed on low molecular weight sulfur species showed that sulfide was produced from trisulfide compounds such as glutathione trisulfide and diallyl trisulfide, but not from the thiol compounds such as cysteine, cystine and glutathione. In the case of plasma proteins, this novel combined detection assay revealed that approximately 14.7, 1.7, 3.9, 3.7 sulfide mol/mol released from human serum albumin, α1-anti-trypsin, α1-acid glycoprotein and ovalbumin, respectively, suggesting that serum albumin is a major pool of polysulfide in human blood circulation. Taken together with the results of albumins of different species, the liberated sulfide has a good correlation with cysteine instead of methionine, indicating the site of incorporation of polysulfide is cysteine. With this novel sulfide detention assay, approximately 8,000, 120 and 1100 µM of polysulfide concentrations was quantitated in human healthy plasma, saliva and tear, respectively. Our promising polysulfide specific detection assay can be a very important tool because quantitative determination of polysulfide sheds light on the functional consequence of protein-bound cysteine polysulfide and expands the research area of reactive oxygen to reactive polysulfide species.

Human serum albumin hydropersulfide is a potent reactive oxygen species scavenger in oxidative stress conditions such as chronic kidney disease.

Recently, hydropersulfide (RSSH) was found to exist in mammalian tissues and fluids. Cysteine hydropersulfide can be found in free cysteine residues as well as in proteins, and it has potent antioxidative activity. Human serum albumin (HSA) is the most abundant protein in mammalian serum. HSA possesses a free thiol group in Cys-34 that could be a site for hydropersulfide formation. HSA hydropersulfide of high purity as a positive control was prepared by treatment of HSA with Na2S. The presence of HSA hydropersulfide was confirmed by spectroscopy and ESI-TOFMS analysis where molecular weights of HSA hydropersulfide by increments of approximately 32 Da (Sulfur atom) were detected. The fluorescent probe results showed that Alexa Fluor 680 conjugated maleimide (Red-Mal) was a suitable assay and bromotrimethylammoniumbimane bromide appeared to be a selective reagent for hydropersulfide. The effect of oxidative stress related disease on the existence of albumin hydropersulfides was examined in rat 5/6 nephrectomy model of chronic kidney disease (CKD). Interestingly, the level of hydropersulfides in rat 5/6 nephrectomy model serum was decreased by a uremic toxin that increases oxidative stress in rat 5/6 nephrectomy model. Furthermore, we demonstrated that the levels of HSA hydropersulfide in human subjects were reduced in CKD but restored by hemodialysis using Red-Mal assay. We conclude that HSA hydropersulfide could potentially play an important role in biological anti-oxidative defense, and it is a promising diagnostic and therapeutic marker of oxidative diseases.

Preimplantation genetic diagnosis/screening by comprehensive molecular testing.

Although embryo screening by preimplantation genetic diagnosis (PGD) has become the standard technique for the treatment of recurrent pregnancy loss in couples with a balanced gross chromosomal rearrangement, the implantation and pregnancy rates of PGD using conventional fluorescence in situ hybridization (FISH) remain suboptimal. Comprehensive molecular testing, such as array comparative genomic hybridization and next-generation sequencing, can improve these rates, but amplification bias in the whole genome amplification method remains an obstacle to accurate diagnosis. Recent advances in amplification procedures combined with improvements in the microarray platform and analytical method have overcome the amplification bias, and the data accuracy of the comprehensive PGD method has reached the level of clinical laboratory testing. Currently, comprehensive PGD is also applied to recurrent pregnancy loss due to recurrent fetal aneuploidy or infertility with recurrent implantation failure, known as preimplantation genetic screening. However, there are still numerous problems to be solved, including misdiagnosis due to somatic mosaicism, cell cycle-related background noise, and difficulty in diagnosis of polyploidy. The technology for comprehensive PGD also requires further improvement.