Engineered Extracellular Vesicles for Delivery of an IL-1 Receptor Antagonist Promote Targeted Repair of Retinal Degeneration.

Retinal degeneration (RD) is an irreversible blinding disease that seriously affects patients' daily activities and mental health. Targeting hyperactivated microglia and regulating polarization are promising strategies for treating the disease. Mesenchymal stem cell (MSC) transplantation is proven to be an effective treatment due to its immunomodulatory and regenerative properties. However, the low efficiency of cell migration and integration of MSCs remains a major obstacle to clinical use. The goal of this study is to develop a nanodelivery system that targets hyperactivated microglia and inhibits their release of proinflammatory factors, to achieve durable neuroprotection. This approach is to engineer extracellular vesicles (EVs) isolated from MSC, modify them with a cyclic RGD (cRGD) peptide on their surface, and load them with an antagonist of the IL-1 receptor, anakinra. Comparing with non-engineered EVs, it is observed that engineered cRGD-EVs exhibit an increased targeting efficiency against hyperactivated microglia and strongly protected photoreceptors in experimental RD cells and animal models. This study provides a strategy to improve drug delivery to degenerated retinas and offers a promising approach to improve the treatment of RD through targeted modulation of the immune microenvironment via engineered cRGD-EVs.

cGAS promotes sepsis in radiotherapy of cancer by up-regulating caspase-11 signaling.

Radiotherapy is the most common strategy in the treatment of cancer. However, radiation-induced acute complications, in particular sepsis, render patients in a life-threatening status or lead to delay of therapy that largely influences patients' overall responses. The understanding of sepsis in radiotherapy is currently scant and effective medicine is not available by far. Here, with WT mice as control, we challenged mice deficient to cGas, Caspase-11, Gsdmd or Asc with cecal ligation and puncture (CLP, a sepsis model) after a treatment of thorax irradiation. We found that radiation robustly upgraded caspase-11 pathway in irradiated region and consequently deteriorated lung injury and mortality in the sepsis model. cGas knockout markedly attenuated radiation-upgraded caspase-11 and restored sepsis. Deficiency of non-canonical inflammasome, caspase-11 and the downstream GSDMD, rather than an AIM2 inflammasome component, ASC, dramatically protected against radiation-promoted injury and mortality in septic mice. The protection may attribute to the inhibition of caspase-11-mediated pyroptosis in endothelial cells of the lung. Thus, blocking cGAS/caspase-11 signaling would be an adjuvant treatment strategy for preventing sepsis in radiotherapy of cancer.

Keratin Profiling by Single-Cell RNA-Sequencing Identifies Human Prostate Stem Cell Lineage Hierarchy and Cancer Stem-Like Cells.

Single prostate stem cells can generate stem and progenitor cells to form prostaspheres in 3D culture. Using a prostasphere-based label retention assay, we recently identified keratin 13 (KRT13)-enriched prostate stem cells at single-cell resolution, distinguishing them from daughter progenitors. Herein, we characterized the epithelial cell lineage hierarchy in prostaspheres using single-cell RNA-seq analysis. Keratin profiling revealed three clusters of label-retaining prostate stem cells; cluster I represents quiescent stem cells (PSCA, CD36, SPINK1, and KRT13/23/80/78/4 enriched), while clusters II and III represent active stem and bipotent progenitor cells (KRT16/17/6 enriched). Gene set enrichment analysis revealed enrichment of stem and cancer-related pathways in cluster I. In non-label-retaining daughter progenitor cells, three clusters were identified; cluster IV represents basal progenitors (KRT5/14/6/16 enriched), while clusters V and VI represent early and late-stage luminal progenitors, respectively (KRT8/18/10 enriched). Furthermore, MetaCore analysis showed enrichment of the "cytoskeleton remodeling-keratin filaments" pathway in cancer stem-like cells from human prostate cancer specimens. Along with common keratins (KRT13/23/80/78/4) in normal stem cells, unique keratins (KRT10/19/6C/16) were enriched in cancer stem-like cells. Clarification of these keratin profiles in human prostate stem cell lineage hierarchy and cancer stem-like cells can facilitate the identification and therapeutic targeting of prostate cancer stem-like cells.

The role of WNT10B in normal prostate gland development and prostate cancer.

BACKGROUND: WNT signaling is implicated in embryonic development, and in adult tissue homeostasis, while its deregulation is evident in disease. This study investigates the unique roles of canonical WNT10B in both normal prostate development and prostate cancer (PCa) progression. METHODS: Organ culture and rat ventral prostates (VPs) were used to study Wnt10b ontogeny and growth effect of WNT10B protein. PB-SV40 LTag rat VPs were utilized for Wnt expression polymerase chain reaction (PCR) array and immunohistochemistry. Human localized PCa tissue microarrays (TMAs) were investigated for differential WNT10B expression. Human RNA-seq data sets were queried for differential expression of WNT10B in metastatic and localized PCa. Knockdown of WNT10B in PC3 cells was utilized to study its effects on proliferation, stemness, epithelial to mesenchymal transition (EMT), and xenograft propagation. RESULTS: Wnt10b expression was highest at birth and rapidly declined in the postnatal rat VP. Exogenous WNT10B addition to culture developing VPs decreased growth suggesting an antiproliferative role. VPs from PB-SV40 LTag rats with localized PCa showed a 25-fold reduction in Wnt10b messenger RNA (mRNA) expession, confirmed at the protein level. Human PCa TMAs revealed elevated WNT10B protein in prostate intraepithelial neoplasia compared with normal prostates but reduced levels in localized PCa specimens. In contrast, RNA-seq data set of annotated human PCa metastasis found a significant increase in WNT10B mRNA expression compared with localized tumors suggesting stage-specific functions of WNT10B. Similarly, WNT10B mRNA levels were increased in metastatic cell lines PC3, PC3M, as well as in HuSLC, a PCa stem-like cell line, as compared with disease-free primary prostate epithelial cells. WNT10B knockdown in PC3 cells reduced expression of EMT genes, MMP9 and stemness genes NANOG and SOX2 and markedly reduced the stem cell-like side population. Furthermore, loss of WNT10B abrogated the ability of PC3 cells to propagate tumors via serial transplantation. CONCLUSIONS: Taken together, these results suggest a dual role for WNT10B in normal development and in PCa progression with opposing functions depending on disease stage. We propose that decreased WNT10B levels in localized cancer allow for a hyperproliferative state, whereas increased levels in advanced disease confer a stemness and malignant propensity which is mitigated by knocking down WNT10B levels. This raises the potential for WNT10B as a novel target for therapeutic intervention in metastatic PCa.

Targeting prostate cancer cells with enzalutamide-HDAC inhibitor hybrid drug 2-75.

BACKGROUND: The progression of castration-resistant prostate cancer (CRPC) still relies on the function of androgen receptor (AR), achieved by evolving mechanisms to reactivate AR signaling under hormonal therapy. Histone deacetylase inhibitors (HDACis) disrupt cytoplasmic AR chaperone heat shock protein 90 (Hsp90) via HDAC6 inhibition, leading to AR degradation and growth suppression of prostate cancer (PCa) cells. However, current HDACis are not effective in clinical trials treating CRPC. METHODS: We designed hybrid molecules containing partial chemical scaffolds of AR antagonist enzalutamide (Enz) and HDACi suberoylanilide hydroxamic acid (SAHA) as new anti-PCa agents. We previously demonstrated that Enz-HDACi hybrid drug 2-75 targets both AR and Hsp90, which inhibits the growth of Enz-resistant C4-2 cells. In the current study, we further investigate the molecular and cellular actions of 2-75 and test its anti-PCa effects in vivo. RESULTS: Compared with Enz, 2-75 had greater AR antagonistic effects by decreasing the stability, transcriptional activity, and nuclear translocation of intracellular AR. In addition to inhibition of full-length AR (FL AR), 2-75 downregulated the AR-V7 variant in multiple PCa cell lines. Mechanistic studies indicated that the AR affinity of 2-75 retains the drug in the cytoplasm of AR + PCa cells and further directs 2-75 to the AR-associated protein complex, which permits localized effects on AR-associated Hsp90. Further, unlike pan-HDACi SAHA, the cytoplasm-retaining property allows 2-75 to significantly inhibit cytoplasmic HDAC6 with limited impact on nuclear HDACs. These selective cytoplasmic actions of 2-75 overcome the unfavorable resistance and toxicity properties associated with classical AR antagonists, HDACis, and Hsp90 inhibitors. Finally, 2-75 showed greater antitumor activities than Enz in vivo on SQ xenografts derived from LNCaP cells. CONCLUSIONS: Novel therapeutic strategy using newly designed 2-75 and related AR antagonist-HDACi hybrid drugs has great potential for effective treatment of CRPC.

Deregulation of Regulatory T Cells in Acute-on-Chronic Liver Failure: A Rat Model.

Aims. Acute-on-chronic liver failure (ACLF) and acute liver failure (ALF) are similar in many respects during their acute exacerbation; however, ACLF generally has a poorer prognosis. We aimed to investigate the role and dynamic changes of regulatory T cell (Treg) and T helper 17 (Th17) cell proportions during ACLF progress. Methods. All rats were classified into two groups randomly: ACLF group and ALF group (control group). The rat model of ACLF was preestablished by intraperitoneal injection of carbon tetrachloride for 2 months. Then acute liver injury was induced by combined D-galactosamine and lipopolysaccharide. Six time points were examined before or after acute induction. Liver samples were performed with hematoxylin-eosin and Masson staining; circulatory Treg and Th17 cell frequencies were determined using flow cytometry assays; serum levels of alanine aminotransferase, aspartate aminotransferase, interleukin-10 (IL-10), and interferon-γ (IFN-γ) were examined. Results. In group ACLF, both Th17 cell proportion and IFN-γ level presented upgrade firstly and then descend latter tendency; the trends of Treg cell proportion and IL-10 level were observed to gradually decrease and became stable. Conclusion. The Treg cells played an important role in the immunologic mechanism during the process of ACLF. And the function of Treg cells in ACLF was defective.

N -methyl- N -nitrosourea-induced retinal degeneration in mice.

Mouse retinal degeneration models have been investigated for many years in the hope of understanding the mechanism of photoreceptor cell death. N -methyl- N -nitrosourea (MNU) has been previously shown to induce outer retinal degeneration in mice. After MNU was intraperitoneally injected in C57/BL mice, we observed a gradual decrease in the outer nuclear layer (ONL) thickness associated with photoreceptor outer segment loss, bipolar cell dendritic retraction and reactive gliosis. Reactive gliosis was confirmed by increased GFAP protein levels. More serious damage to the central retina as opposed to the peripheral retina was found in the MNU-induced retinal degeneration model. Retinal ganglion cells (RGC) appear to be spared for at least two months after MNU treatment. Following retinal vessel labelling, we observed vascular complexes in the distal vessels, indicating retinal vessel damage. In the remnant retinal photoreceptor of the MNU-treated mouse, concentrated colouring nuclei were detected by electron microscopy, together with the loss of mitochondria and displaced remnant synaptic ribbons in the photoreceptor. We also observed decreased mitochondrial protein levels and increased amounts of nitrosylation/nitration in the photoreceptors. The mechanism of MNU-induced apoptosis may result from oxidative stress or the loss of retinal blood supply. MNU-induced mouse retinal degeneration in the outer retina is a useful animal model for photoreceptor degeneration diseases, such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP).

Complex in vitro Model: A Transformative Model in Drug Development and Precision Medicine.

In vitro and in vivo models play integral roles in preclinical drug research, evaluation, and precision medicine. In vitro models primarily involve research platforms based on cultured cells, typically in the form of two-dimensional (2D) cell models. However, notable disparities exist between 2D cultured cells and in vivo cells across various aspects, rendering the former inadequate for replicating the physiologically relevant functions of human or animal organs and tissues. Consequently, these models failed to accurately reflect real-life scenarios post-drug administration. Complex in vitro models (CIVMs) refer to in vitro models that integrate a multicellular environment and a three-dimensional (3D) structure using bio-polymer or tissue-derived matrices. These models seek to reconstruct the organ- or tissue-specific characteristics of the extracellular microenvironment. The utilization of CIVMs allows for enhanced physiological correlation of cultured cells, thereby better mimicking in vivo conditions without ethical concerns associated with animal experimentation. Consequently, CIVMs have gained prominence in disease research and drug development. This review aimed to comprehensively examine and analyze the various types, manufacturing techniques, and applications of CIVM in the domains of drug discovery, drug development, and precision medicine. The objective of this study was to provide a comprehensive understanding of the progress made in CIVMs and their potential future use in these fields.

The preclinical and clinical progress of cell sheet engineering in regenerative medicine.

Cell therapy is an accessible method for curing damaged organs or tissues. Yet, this approach is limited by the delivery efficiency of cell suspension injection. Over recent years, biological scaffolds have emerged as carriers of delivering therapeutic cells to the target sites. Although they can be regarded as revolutionary research output and promote the development of tissue engineering, the defect of biological scaffolds in repairing cell-dense tissues is apparent. Cell sheet engineering (CSE) is a novel technique that supports enzyme-free cell detachment in the shape of a sheet-like structure. Compared with the traditional method of enzymatic digestion, products harvested by this technique retain extracellular matrix (ECM) secreted by cells as well as cell-matrix and intercellular junctions established during in vitro culture. Herein, we discussed the current status and recent progress of CSE in basic research and clinical application by reviewing relevant articles that have been published, hoping to provide a reference for the development of CSE in the field of stem cells and regenerative medicine.

Stem cells from a malignant rat prostate cell line generate prostate cancers in vivo: a model for prostate cancer stem cell propagated tumor growth.

Cancer stem cells (CSCs) are resistant to conventional cancer therapies, permitting the repopulation of new tumor growth and driving disease progression. Models for testing prostate CSC-propagated tumor growth are presently limited yet necessary for therapeutic advancement. Utilizing the congenic nontumorigenic NRP152 and tumorigenic NRP154 rat prostate epithelial cell lines, the present study investigated the self-renewal, differentiation, and regenerative abilities of prostate stem/progenitor cells and developed a CSC-based PCa model. NRP154 cells expressed reduced levels of tumor suppressor caveolin-1 and increased p-Src as compared to NRP152 cells. Gene knockdown of caveolin-1 in NRP152 cells upregulated p-Src, implicating their role as potential oncogenic mediators in NRP154 cells. A FACS-based Hoechst exclusion assay revealed a side population of stem-like cells (0.1%) in both NRP152 and NRP154 cell lines. Using a 3D Matrigel culture system, stem cells from both cell lines established prostaspheres at a 0.1% efficiency through asymmetric self-renewal and rapid proliferation of daughter progenitor cells. Spheres derived from both cell lines contained CD117+ and CD133+ stem cell subpopulations and basal progenitor cell subpopulations (p63+ and CK5+) but were negative for luminal cell CK8 markers at day 7. While some NRP152 sphere cells were androgen receptor (AR) positive at this timepoint, NRP154 cells were AR- up to 30 days of 3D culture. The regenerative capacity of the stem/progenitor cells was demonstrated by in vivo tissue recombination with urogenital sinus mesenchyme (UGM) and renal grafting in nude mice. While stem/progenitor cells from NRP152 spheroids generated normal prostate structures, CSCs and progeny cells from NRP154 tumoroids generated tumor tissues that were characterized by immunohistochemistry. Atypical hyperplasia and prostatic intraepithelial neoplasia (PIN) lesions progressed to adenocarcinoma with kidney invasion over 4 months. This provides clear evidence that prostate CSCs can repopulate new tumor growth outside the prostate gland that rapidly progresses to poorly differentiated adenocarcinoma with invasive capabilities. The dual in vitro/in vivo CSC model system presented herein provides a novel platform for screening therapeutic agents that target prostate CSCs for effective combined treatment protocols for local and advanced disease stages.

Design and synthesis of isothiocyanate-containing hybrid androgen receptor (AR) antagonist to downregulate AR and induce ferroptosis in GSH-Deficient prostate cancer cells.

Sustained androgen receptor (AR) signaling and apoptosis evasion are among the main hurdles of castration-resistant prostate cancer (CRPC) treatment. We designed and synthesized isothiocyanate (ITC)-containing hybrid AR antagonist (ITC-ARi) and rationally combined ITC-ARi with GSH synthesis inhibitor buthionine sulfoximine (BSO) to efficiently downregulate AR/AR splice variant and induce ferroptosis in CRPC cells. The representative ITC-ARi 13 is an AR ligand that contains an N-acetyl cysteine-masked ITC moiety and gradually releases parental unconjugated ITC 12b in aqueous solution. The in vitro anti-PCa activities of 13, such as growth inhibition and AR downregulation, are significantly enhanced when combined with BSO. The drug combination caused notable lipid peroxidation and the cell viability was effectively rescued by iron chelator, antioxidants or the inhibitor of heme oxygenase-1, supporting the induction of ferroptosis. 13 and BSO cooperatively downregulate AR and induce ferroptosis likely through increasing the accessibility of 13/12b to cellular targets, escalating free intracellular ferrous iron and attenuating GSH-centered cellular defense and adaptation. Further studies on the combination of ITC-ARi and GSH synthesis inhibitor could result in a new modality against CRPC.

Aberrant Methylation and Differential Expression of SLC2A1, TNS4, GAPDH, ATP8A2, and CASZ1 Are Associated with the Prognosis of Lung Adenocarcinoma.

Lung cancer is one of the leading triggers for cancer death worldwide. In this study, the relationship of the aberrantly methylated and differentially expressed genes in lung adenocarcinoma (LUAD) with cancer prognosis was investigated, and 5 feature genes were identified eventually. Specifically, we firstly downloaded the LUAD-related mRNA expression profile (including 57 normal tissue samples and 464 LUAD tissue samples) and Methy450 expression data (including 32 normal tissue samples and 373 LUAD tissue samples) from the TCGA database. The package "limma" was used to screen differentially expressed genes and aberrantly methylated genes, which were intersected for identifying the hypermethylated downregulated genes (DGs Hyper) and the hypomethylated upregulated genes (UGs Hypo). GO annotation and KEGG pathway enrichment analysis were further performed, and it was found that these DGs Hyper and UGs Hypo were predominantly activated in the biological processes and signaling pathways such as the regulation of vasculature development, DNA-binding transcription activator activity, and Ras signaling pathway, indicating that these genes play a vital role in the initiation and progression of LUAD. Additionally, univariate and multivariate Cox regression analyses were conducted to find the genes significantly associated with LUAD prognosis. Five genes including SLC2A1, TNS4, GAPDH, ATP8A2, and CASZ1 were identified, with the former three highly expressed and the latter two poorly expressed in LUAD, indicating poor prognosis of LUAD patients as judged by survival analysis.

Effects of Inorganic Arsenic on Human Prostate Stem-Progenitor Cell Transformation, Autophagic Flux Blockade, and NRF2 Pathway Activation.

BACKGROUND: Inorganic arsenic (iAs) is an environmental toxicant associated with an increased risk of prostate cancer in chronically exposed populations worldwide. However, the biological mechanisms underlying iAs-induced prostate carcinogenesis remain unclear. OBJECTIVES: We studied how iAs affects normal human prostate stem-progenitor cells (PrSPCs) and drives transformation and interrogated the molecular mechanisms involved. METHODS: PrSPCs were enriched by spheroid culture from normal human primary or immortalized prostate epithelial cells, and their differentiation capability was evaluated by organoid culture. Microarray analysis was conducted to identify iAs-dysregulated genes, and lentiviral infection was used for stable manipulation of identified genes. Soft agar colony growth assays were applied to examine iAs-induced transformation. For in vivo study, PrSPCs mixed with rat urogenital sinus mesenchyme were grafted under the renal capsule of nude mice to generate prostatelike tissues, and mice were exposed to 5 ppm (∼65µM) iAs in drinking water for 3 months. RESULTS: Low-dose iAs (1µM) disturbed PrSPC homeostasis in vitro, leading to increased self-renewal and suppressed differentiation. Transcriptomic analysis indicated that iAs activated oncogenic pathways in PrSPCs, including the KEAP1-NRF2 pathway. Further, iAs-exposed proliferative progenitor cells exhibited NRF2 pathway activation that was sustained in their progeny cells. Knockdown of NRF2 inhibited spheroid formation by driving PrSPC differentiation, whereas its activation enhanced spheroid growth. Importantly, iAs-induced transformation was suppressed by NRF2 knockdown. Mechanistically, iAs suppressed Vacuolar ATPase subunit VMA5 expression, impairing lysosome acidification and inhibiting autophagic protein degradation including p62, which further activated NRF2. In vivo, chronic iAs exposure activated NRF2 in both epithelial and stroma cells of chimeric human prostate grafts and induced premalignant events. CONCLUSIONS: Low-dose iAs increased self-renewal and decreased differentiation of human PrSPCs by activating the p62-NRF2 axis, resulting in epithelial cell transformation. NRF2 is activated by iAs through specific autophagic flux blockade in progenitor cells, which may have potential therapeutic implications. https://doi.org/10.1289/EHP6471.

Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures.

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident stem cells are relatively quiescent with niche restraints in adult tissues, they enter the cell cycle in anchor-free three-dimensional (3D) culture and undergo both symmetric and asymmetric cell division, giving rise to both stem and progenitor cells. The latter proliferate rapidly and are the major cell population at various stages of lineage commitment, forming heterogeneous spheroids. Using primary normal human prostate epithelial cells (HPrEC), a spheroid-based, label-retention assay was developed that permits the identification and functional isolation of the spheroid-initiating stem cells at a single cell resolution. HPrEC or cell lines are two-dimensionally (2D) cultured with BrdU for 10 days to permit its incorporation into the DNA of all dividing cells, including self-renewing stem cells. Wash out commences upon transfer to the 3D culture for 5 days, during which stem cells self-renew through asymmetric division and initiate spheroid formation. While relatively quiescent daughter stem cells retain BrdU-labeled parental DNA, the daughter progenitors rapidly proliferate, losing the BrdU label. BrdU can be substituted with CFSE or Far Red pro-dyes, which permit live stem cell isolation by FACS. Stem cell characteristics are confirmed by in vitro spheroid formation, in vivo tissue regeneration assays, and by documenting their symmetric/asymmetric cell divisions. The isolated label-retaining stem cells can be rigorously interrogated by downstream molecular and biologic studies, including RNA-seq, ChIP-seq, single cell capture, metabolic activity, proteome profiling, immunocytochemistry, organoid formation, and in vivo tissue regeneration. Importantly, this marker-free functional stem cell isolation approach identifies stem-like cells from fresh cancer specimens and cancer cell lines from multiple organs, suggesting wide applicability. It can be used to identify cancer stem-like cell biomarkers, screen pharmaceuticals targeting cancer stem-like cells, and discover novel therapeutic targets in cancers.

Differential Actions of Estrogen Receptor α and ß via Nongenomic Signaling in Human Prostate Stem and Progenitor Cells.

Human prostate stem and progenitor cells express estrogen receptor (ER)α and ERß and exhibit proliferative responses to estrogens. In this study, membrane-initiated estrogen signaling was interrogated in human prostate stem/progenitor cells enriched from primary epithelial cultures and stem-like cell lines from benign and cancerous prostates. Subcellular fractionation and proximity ligation assays localized ERα and ERß to the cell membrane with caveolin-1 interactions. Exposure to 17ß-estradiol (E2) for 15 to 60 minutes led to sequential phosphorylation of signaling molecules in MAPK and AKT pathways, IGF1 receptor, epidermal growth factor receptor, and ERα, thus documenting an intact membrane signalosome that activates diverse downstream cascades. Treatment with an E2-dendrimer conjugate or ICI 182,870 validated E2-mediated actions through membrane ERs. Overexpression and knockdown of ERα or ERß in stem/progenitor cells identified pathway selectivity; ERα preferentially activated AKT, whereas ERß selectively activated MAPK cascades. Furthermore, prostate cancer stem-like cells expressed only ERß, and brief E2 exposure activated MAPK but not AKT cascades. A gene subset selectively regulated by nongenomic E2 signaling was identified in normal prostate progenitor cells that includes BGN, FOSB, FOXQ1, and MAF. Membrane-initiated E2 signaling rapidly modified histone methyltransferases, with MLL1 cleavage observed downstream of phosphorylated AKT and EZH2 phosphorylation downstream of MAPK signaling, which may jointly modify histones to permit rapid gene transcription. Taken together, the present findings document ERα and ERß membrane-initiated signaling in normal and cancerous human prostate stem/progenitor cells with differential engagement of downstream effectors. These signaling pathways influence normal prostate stem/progenitor cell homeostasis and provide novel therapeutic sites to target the elusive prostate cancer stem cell population.

Hypoxia­inducible factor 1α and ROCK1 regulate proliferation and collagen synthesis in hepatic stellate cells under hypoxia.

Hypoxia serves a critical role in the pathogenesis of liver fibrosis. Hypoxia­inducible factor 1α (HIF1­α) is induced when cells are exposed to low O2 concentrations. Recently, it has been suggested that Rho­associated coiled­coil­forming kinase 1 (ROCK1) may be an important HIF1­α regulator. In the present study, it was analyzed whether crosstalk between HIF1­α and ROCK1 regulates cell proliferation and collagen synthesis in hepatic stellate cells (HSCs) under hypoxic conditions. For this purpose, a rat hepatic HSC line (HSC­T6) was cultured under hypoxic or normoxic conditions, and HIF1­α and ROCK1 expression was measured at different time points. Additionally, HSC­T6 cells were transfected with HIF1­α small interfering RNA (siHIF1­α), and measured protein expression and mRNA transcript levels of α­smooth muscle actin, collagen 1A1 and ROCK1. Collagen 3A1 secretion was also measured by ELISA. Cell proliferation was assessed by the MTT assay under these hypoxic conditions. The results indicated that a specific ROCK inhibitor, Y­27632, increased HIF1­α and ROCK1 expression over time in HSC­T6 cells in response to hypoxia. In addition, knockdown of HIF1­α inhibited HSC­T6 proliferation, suppressed collagen 1A1 expression, decreased collagen 3A1 secretion and attenuated ROCK1 expression. Notably, ROCK1 inhibition caused HSC­T6 quiescence, suppressed collagen secretion and downregulated HIF1­α expression. Collectively, these findings indicated that the interplay between HIF1­α and ROCK1 may be a critical factor that regulates cell proliferation and collagen synthesis in rat HSCs under hypoxia.

Evaluation of Bisphenol A (BPA) Exposures on Prostate Stem Cell Homeostasis and Prostate Cancer Risk in the NCTR-Sprague-Dawley Rat: An NIEHS/FDA CLARITY-BPA Consortium Study.

BACKGROUND: Previous work determined that early life exposure to low-dose Bisphenol A (BPA) increased rat prostate cancer risk with aging. Herein, we report on prostate-specific results from CLARITY-BPA (Consortium Linking Academic and Regulatory Insights on BPA Toxicity), which aims to resolve uncertainties regarding BPA toxicity. OBJECTIVES: We sought to a) reassess whether a range of BPA exposures drives prostate pathology and/or alters prostatic susceptibility to hormonal carcinogenesis, and b) test whether chronic low-dose BPA targets prostate epithelial stem and progenitor cells. METHODS: Sprague-Dawley rats were gavaged daily with vehicle, ethinyl estradiol (EE) or [Formula: see text] BPA/kg-BW during development or chronically, and prostate pathology was assessed at one year. One developmentally exposed cohort was given testosterone plus estradiol ([Formula: see text]) implants at day 90 to promote carcinogenesis with aging. Epithelial stem and progenitor cells were isolated by prostasphere (PS) culture from dorsolateral prostates (DLP) of rats continuously exposed for six months to [Formula: see text] BPA/kg-BW. Gene expression was analyzed by quantitative real time reverse transcription polymerase chain reaction (qRT-PCR). RESULTS: Exposure to BPA alone at any dose did not drive prostate pathology. However, rats treated with EE, 2.5, 250, or [Formula: see text] BPA/kg-BW plus [Formula: see text] showed greater severity of lateral prostate intraepithelial neoplasia (PIN), and DLP ductal adenocarcinoma multiplicity was markedly elevated in tumor-bearing rats exposed to [Formula: see text]-BW. DLP stem cells, assessed by PS number, doubled with chronic EE and [Formula: see text] exposures. PS size, reflecting progenitor cell proliferation, was greater at 25 and [Formula: see text] BPA doses, which also shifted lineage commitment toward basal progenitors while reducing luminal progenitor cells. CONCLUSIONS: Together, these results confirm and extend previous evidence using a rat model and human prostate epithelial cells that low-dose BPA augments prostate cancer susceptibility and alters adult prostate stem cell homeostasis. Therefore, we propose that BPA exposures may contribute to the increased carcinogenic risk in humans that occurs with aging. https://doi.org/10.1289/EHP3953.

Correlation between high mobility group box-1 protein and chronic hepatitis B infection with severe hepatitis B and acute-on-chronic liver failure: a meta-analysis.

INTRODUCTION: The aim of this study was to evaluate the correlation of High-mobility group box 1 (HMGB1) expression in the serum with chronic hepatitis B (CHB) related liver fibrosis, severe hepatitis B and acute-on-chronic liver failure (ACLF). EVIDENCE ACQUISITION: We made a literature search in PubMed, Embase, Web of Science, Medline, Google Scholar, China National Knowledge Infrastructure, WanFang with no language restriction. Pooled data were analyzed and mean difference with corresponding 95% confidence intervals were calculated. EVIDENCE SYNTHESIS: A total of 16 relevant studies were identified. HMGB1 serum levels were higher in severe hepatitis B or ACLF patients than those in CHB patients. Pooled mean differences of HMGB1 in severe hepatitis B and ACLF patients compared with CHB patients were 4.32 (95% CI: 0.34-8.29, Z=2.13, I2=59%, P=0.03) and 15.96 (95% CI: -0.37-32.28, Z=1.92, P=0.06). Four studies showed there was a different HMGB1 expression in mild, moderate and severe CHB patients (P values were <0.05, <0.05, <0.05 and <0.01, respectively). Pooled mean difference of HMGB1 in low liver fibrosis patients compared with high liver fibrosis was -125.38 (95% CI: -539.44-288.68, Z=0.59, I2=98%, P=0.55). CONCLUSIONS: The results suggested that HMGB1 levels in the serum were statistically higher in severe hepatitis B and ACLF patients. Therefore, HMGB1 may be a useful therapeutic target for severe hepatitis B and ACLF diagnosis.

miR­21 promotes α­SMA and collagen I expression in hepatic stellate cells via the Smad7 signaling pathway.

The aim of the present study was to investigate the role of microRNA (miR)­21 in regulating collagen I and Smad7 expression in activated rat hepatic stellate cells (HSCs). Rat HSCs were isolated by single­step density gradient centrifugation with Nycodenz. Cellular content of miR­21, SMAD7, α­smooth muscle actin (α­SMA), collagen type I alpha 1 (COLLA1) and COLL alpha 2 (A2) mRNA was examined by reverse transcription­quantitative polymerase chain reaction (RT­qPCR), and cellular content of Smad7 and α­SMA protein was detected by western blotting. Binding of miR­21 to the 3'­untranslated region (UTR) of Smad7 was verified by dual­luciferase assay. The authors observed that, in activated HSCs, expression of miR­21 was significantly increased in a time­dependent manner, while expression of Smad7 mRNA and protein was significantly reduced. In addition, miR­21 mimics significantly enhanced cellular α­SMA mRNA and protein content, while miR­21 inhibitor significantly reduced α­SMA mRNA and protein levels. Similarly, cellular content of COLLA1 and COLLA2 mRNA was significantly elevated by miR­21 mimics, but reduced by miR­21 inhibitor, in activated HSCs. Moreover, cellular content of Smad7 mRNA and protein was significantly reduced by miR­21 mimics, but significantly increased by miR­21 inhibitor. Furthermore, miR­21 mimics activated firefly luciferase in HEK293 cells transfected with the wild type 3'­UTR of Smad7. miR­21 regulates expression of α­SMA and collagen I in activated rat HSCs by directly targeting Smad7.