The development of early human lymphatic vessels as characterized by lymphatic endothelial markers.

Lymphatic vessel development studies in mice and zebrafish models have demonstrated that lymphatic endothelial cells (LECs) predominantly differentiate from venous endothelial cells via the expression of the transcription factor Prox1. However, LECs can also be generated from undifferentiated mesoderm, suggesting potential diversity in their precursor cell origins depending on the organ or anatomical location. Despite these advances, recapitulating human lymphatic malformations in animal models has been difficult, and considering lymphatic vasculature function varies widely between species, analysis of development directly in humans is needed. Here, we examined early lymphatic development in humans by analyzing the histology of 31 embryos and three 9-week-old fetuses. We found that human embryonic cardinal veins, which converged to form initial lymph sacs, produce Prox1-expressing LECs. Furthermore, we describe the lymphatic vessel development in various organs and observe organ-specific differences. These characterizations of the early development of human lymphatic vessels should help to better understand the evolution and phylogenetic relationships of lymphatic systems, and their roles in human disease.

Supplementation with autologous adipose stem cell-derived mitochondria can be a safe and promising strategy for improving oocyte quality.

PURPOSE: In our previous study, we confirmed that the supplementation of vitrified-warmed murine oocytes with autologous adipose stem cell (ASC)-derived mitochondria during intracytoplasmic sperm injection enhances post-fertilization developmental competence in mice. To ensure the safety of this technology, we conducted a thorough study in mice to investigate the potential presence of specific malformations in offspring developed from this approach. METHODS: A transgenerational comparative analysis was conducted on founder mice from embryos that developed after mitochondrial supplementation, and two subsequent generations. Reproductive performance, body growth rate, histopathological parameters, hematological parameters, daily activity patterns, and daily body temperature changes in male and female mice across these three generations were assessed in comparison to wild-type mice of the same age. RESULTS: Both male and female animals in all three generations showed comparable reproductive performance to the control group. Additionally, body growth rate by the age of 8 weeks were found to be comparable to controls across all three generations. Notably, no significant histopathological abnormalities were detected in vital organs, including the brain, heart, liver, kidneys, lungs, ovaries, and testes, in any individuals from the studied cohorts. The blood parameters were consistent with the control data. The continuous monitoring of activity and body temperature changes (both day and night) over a 1-week period revealed a pattern closely resembling that observed in the control animals. CONCLUSION: Injection of ASC-mitochondria into oocytes may be a promising technique to support developmental potential without causing adverse epigenetic events in the offspring in mice. However, before considering clinical application, additional safety screening using larger animals or non-human primates is essential.

Fibroblast-derived exosomal microRNA regulates NKX3-1 expression in androgen-sensitive, androgen receptor-dependent prostate cancer cells.

Androgen deprivation therapy (ADT) targeting androgen production and androgen receptor (AR) signaling is the primary antihormonal therapy in the treatment of advanced prostate cancer (PCa). However, no clinically established molecular biomarkers have been identified to predict the effectiveness of ADT before starting ADT. The tumor microenvironment of PCa contains fibroblasts that regulate PCa progression by producing multiple soluble factors. We have previously reported that AR-activating factor-secreted fibroblasts increase the responsiveness of androgen-sensitive, AR-dependent PCa cells to ADT. Thus, we hypothesized that fibroblast-derived soluble factors may affect cancer cell differentiation by regulating cancer-related gene expression in PCa cells and that the biochemical characteristics of fibroblasts may be used to predict the effectiveness of ADT. Here, we investigated the effects of normal fibroblasts (PrSC cells) and three PCa patient-derived fibroblast lines (pcPrF-M5, -M28, and -M31 cells) on the expression of cancer-related genes in androgen-sensitive, AR-dependent human PCa cells (LNCaP cells) and three sublines showing different androgen sensitivities and AR dependencies. The mRNA expression of the tumor suppressor gene NKX3-1 in LNCaP cells and E9 cells (which show low androgen sensitivity and AR dependency) was significantly increased by treatment with conditioned media from PrSC and pcPrF-M5 cells but not from pcPrF-M28 and pcPrF-M31 cells. Notably, no upregulation of NKX3-1 was observed in F10 cells (AR-V7-expressing, AR-independent cells with low androgen sensitivity) and AIDL cells (androgen-insensitive, AR-independent cells). Among 81 common fibroblast-derived exosomal microRNAs that showed 0.5-fold lower expression in pcPrF-M28 and pcPrF-M31 cells than in PrSC and pcPrF-M5 cells, miR-449c-3p and miR-3121-3p were found to target NKX3-1. In only LNCaP cells, the NKX3-1 mRNA expression was significantly increased by transfection of an miR-3121-3p mimic but not that of the miR-449c-3p mimic. Thus, fibroblast-derived exosomal miR-3121-3p may be involved in preventing the oncogenic dedifferentiation of PCa cells by targeting NKX3-1 in androgen-sensitive, AR-dependent PCa cells.

Short-term prognosis of low-risk prostate cancer patients is favorable despite the presence of pathological prognostic factors: a retrospective study.

BACKGROUND: Prostate cancer patients with pathological prognostic factors have a poor prognosis, but it is unclear whether pathological prognostic factors are associated with prognosis limited to low-risk patients with good prognosis according to NCCN guidelines. The present study examined whether prognosis is influenced by pathological prognostic factors using radical prostatectomy (RP) specimens from low-risk patients. METHODS: We evaluated diagnostic accuracy by examining biochemical recurrence (BCR)-free survival with respect to clinical and pathological prognostic factors in 419 all-risk patients who underwent RP. Clinical prognostic factors included age, prostate-specific antigen (PSA) levels, PSA density, and risk stratification, while pathological prognostic factors included grade group, lymphovascular space invasion, extraprostatic extension, surgical margins, seminal vesicle invasion, intraductal carcinoma of the prostate (IDCP), and pT. In a subsequent analysis restricted to 104 low-risk patients, survival curves were estimated for pathological prognostic factors using the Kaplan-Meier method and compared using log-rank and generalized Wilcoxon tests. RESULTS: In the overall risk analysis, the presence of pathological prognostic factors significantly shortened BCR-free survival (p < 0.05). Univariable analysis revealed that PSA density, risk categories, and pathological prognostic factors were significantly associated with BCR-free survival, although age and PSA were not. In multivariable analysis, age, risk categories, grade group, IDCP, and pT significantly predicted BCR-free survival (p < 0.05). Conversely, no statistically significant differences were found for any pathological prognostic factors in low-risk patients. CONCLUSIONS: In low-risk patients, pathological prognostic factors did not affect BCR-free survival, which suggests that additional treatment may be unnecessary even if pathological prognostic factors are observed in low-risk patients with RP.

Increased Mortality Risk at Septic Condition in Inflammatory Skin Disorders and the Effect of High-Fat Diet Consumption.

In recent years, attention has increasingly focused on various infectious diseases. Although some fatalities are directly attributed to the causative virus, many result from complications and reactive inflammation. Patients with comorbidities are at a higher risk of mortality. Refractory skin conditions such as atopic dermatitis, psoriasis, and epidermolysis bullosa, known for an elevated risk of sepsis, partly owe this to compromised surface barrier function. However, the detailed mechanisms underlying this phenomenon remain elusive. Conversely, although the detrimental effects of a high-fat diet on health, including the onset of metabolic syndrome, are widely recognized, the association between diet and susceptibility to sepsis has not been extensively explored. In this study, we examined the potential causes and pathogenesis of increased sepsis susceptibility in inflammatory skin diseases using a mouse dermatitis model: keratin 14-driven caspase-1 is overexpressed (KCASP1Tg) in mice on a high-fat diet. Our findings reveal that heightened mortality in the dermatitis mouse model is caused by the inflamed immune system due to the chronic inflammatory state of the local skin, and administration of LPS causes a rapid increase in inflammatory cytokine levels in the spleen. Intake of a high-fat diet exacerbates these cytokine levels. Interestingly, we also observed a reduced expression of Toll-like receptor 4 (TLR4) in monocytes from KCASP1Tg mice, potentially predisposing these animals to heightened infection risks and associated complications. Histological analysis showed a clear decrease in T and B cells in the spleen of KCASP1Tg mice fed a high-fat diet. Thickening of the alveolar wall, inflammatory cell infiltration, and alveolar hemorrhage were more prominent in the lungs of KCASP1Tg and KCASP1Tg with fat mice. We postulate that the chronic, non-infectious inflammation induces a negative feedback loop within the inflammatory cascade, and the suppressed expression of TLR4 renders the mice more susceptible to infections. Therefore, it is imperative for individuals with chronic skin inflammation to closely monitor disease progression upon infection and seek timely and appropriate treatment. Additionally, chronic inflammation of adipose tissue, induced by high-fat food intake, combined with dermatitis inflammation, may exacerbate infections, necessitating a review of dietary habits.

IL-17A Is the Critical Cytokine for Liver and Spleen Amyloidosis in Inflammatory Skin Disease.

Systemic amyloidosis is recognized as a serious complication of rheumatoid arthritis or inflammatory bowel disease, but also of inflammatory skin disease. However, the detailed molecular mechanism of amyloidosis associated with cutaneous inflammation remains unclear, and therapeutic approaches are limited. Here, we investigated the pathophysiology of amyloidosis secondary to cutaneous inflammation and the therapeutic effects of Janus kinase (JAK) inhibitors by examining a mouse model of spontaneous dermatitis (KCASP1Tg mice). Moreover, KCASP1Tg mice were crossed with interleukin-17A (IL-17A) knockout mice to generate IL-17A-/KCASP1Tg and examine the role of IL-17A in amyloidosis under cutaneous inflammation. KCASP1Tg mice showed severe amyloid deposition in the liver and spleen. Increased serum-neutral fat levels and decreased lymphocyte production were observed in the spleen. Overproduction of amyloidosis was partially ameliorated by the administration of JAK inhibitors and was further improved in IL-17A-/KCASP1Tg mice. IL-17A-producing cells included CD4, gamma delta, and CD8 T cells. In summary, our results from the analysis of a mouse model of dermatitis revealed that skin-derived inflammatory cytokines can induce amyloid deposition in the liver and spleen, and that the administration of JAK inhibitors and, even more, IL-17A ablation, reduced amyloidosis. This study demonstrates that active control of skin inflammation is essential to prevent internal organ amyloidosis.

Heterogeneous induction of an invasive phenotype in prostate cancer cells by coculturing with patient-derived fibroblasts.

Prostate cancer (PCa) cells frequently invade the surrounding stroma, leading to heterogeneous formation of structural atypia. The surrounding stroma contains multiple functionally diverse populations of fibroblasts that trigger numerous changes in PCa cells including motility. Thus, we hypothesized that direct or indirect contact of PCa cells with fibroblasts determines an invasive phenotype in PCa cells. We investigated the effects of 10 different patient-derived fibroblast lines on the three-dimensional (3D) morphogenesis of PCa cells growing on a viscous substrate in vitro. When grown alone, all 10 patient-derived fibroblast lines clumped on the viscous substrate, whereas the human androgen-sensitive PCa cell line LNCaP did not. Cocultures of LNCaP cells with seven of the patient-derived fibroblast lines (PrSC, pcPrF-M5, pcPrF-M7, pcPrF-M23, pcPrF-M24, pcPrF-M28, and pcPrF-M31) formed a thick fibroblast layer that resembled human prostate stromal structures. In contrast, cocultures of LNCaP cells with the remaining three fibroblast lines (NPF-M13, pcPrF-M10, and pcPrF-M26) did not form a thick fibroblast layer. Of the seven fibroblast lines that caused thick layer formation, four patient-derived fibroblast lines (PrSC, pcPrF-M5, pcPrF-M28, and pcPrF-M31) induced an invasive phenotype in LNCaP cells with a cord-like infiltrating growth pattern, whereas the other three fibroblast lines (pcPrF-M7, pcPrF-M23, and pcPrF-M24) induced no or a very weak invasive phenotype. Using cell culture inserts, none of the four patient-derived fibroblast lines that induced an invasive phenotype (PrSC, pcPrF-M5, pcPrF-M28, and pcPrF-M31) affected CDH1 mRNA expression in LNCaP cells; yet, two patient-derived fibroblast lines (pcPrF-M5 and pcPrF-M28) increased CDH2 mRNA expression in LNCaP cells, whereas the other two fibroblast lines (PrSC and pcPrF-M31) did not. These results suggest that the existence of multiple functionally diverse populations of fibroblasts in PCa tissue may be responsible for the diversity in PCa cell invasion, leading to heterogeneous formation of structural atypia.

New horizons of DNA adductome for exploring environmental causes of cancer.

Chemical carcinogenesis is focused on the formation of DNA adducts, a form of DNA damage caused by covalent binding of a chemical moiety to DNA. The detection of carcinogen-DNA adducts in human tissues, along with demonstration of mutagenicity/carcinogenicity in experimental systems, and validation of adducts as biomarkers of environmental exposure and indicators of cancer risk in molecular epidemiological studies suggests a pivotal role of DNA adducts in cancer development. However, accurate measurement of DNA adducts in varied biological samples is challenging. Advances in mass spectrometry have prompted the development of DNA adductome analysis, an emerging method that simultaneously screens for multiple DNA adducts and provides relevant structural information. In this review, we summarize the basic principle and applications of DNA adductome analysis that would contribute to the elucidation of the environmental causes of cancer. Based on parallel developments in several fields, including next-generation sequencing, we describe a new approach used to explore cancer etiology, which integrates analyses of DNA adductome data and mutational signatures derived from whole-genome/exome sequencing.

TDP2 suppresses genomic instability induced by androgens in the epithelial cells of prostate glands.

Androgens stimulate the proliferation of epithelial cells in the prostate by activating topoisomerase 2 (TOP2) and regulating the transcription of target genes. TOP2 resolves the entanglement of genomic DNA by transiently generating double-strand breaks (DSBs), where TOP2 homodimers covalently bind to 5' DSB ends, called TOP2-DNA cleavage complexes (TOP2ccs). When TOP2 fails to rejoin TOP2ccs generating stalled TOP2ccs, tyrosyl DNA phosphodiesterase-2 (TDP2) removes 5' TOP2 adducts from stalled TOP2ccs prior to the ligation of the DSBs by nonhomologous end joining (NHEJ), the dominant DSB repair pathway in G0 /G1 phases. We previously showed that estrogens frequently generate stalled TOP2ccs in G0 /G1 phases. Here, we show that physiological concentrations of androgens induce several DSBs in individual human prostate cancer cells during G1 phase, and loss of TDP2 causes a five times higher number of androgen-induced chromosome breaks in mitotic chromosome spreads. Intraperitoneally injected androgens induce several DSBs in individual epithelial cells of the prostate in TDP2-deficient mice, even at 20 hr postinjection. In conclusion, physiological concentrations of androgens have very strong genotoxicity, most likely by generating stalled TOP2ccs.

Castration-induced stromal remodeling disrupts the reconstituted prostate epithelial structure.

The normal prostate epithelial structure is maintained by homeostatic interactions with smooth muscle cells. However, structural alterations of the stroma are commonly observed in prostatic proliferative diseases, leading to the abnormalities of prostate epithelial structure. A decrease in the androgen level experimentally induces stromal remodeling, i.e., replacement of smooth muscle cells with fibroblasts or myofibroblasts. In this study, we investigated the effects of castration-induced stromal remodeling and subsequent aberrant activation of epithelial-stromal interactions on the reconstituted human prostate-like epithelial structure. We performed in vivo experiments using the human prostate epithelial cell line BPH-1 and fetal rat urogenital sinus mesenchyme to generate heterotypic tissue recombinants that form human prostate-like epithelial structure (i.e., solid- and canalized-epithelial cords). Host mice were castrated at 12 weeks post transplantation (castration) and implanted with a dihydrotestosterone pellet at 14 days post castration (androgen replacement treatment; ART). In the castration group, the percentages of fibrotic area and disrupted prostate epithelial structure without the basement membrane (BM) increased proportionally in a time-dependent manner, but were suppressed by ART. In the castration group, tenascin-C (TNC)-positive fibroblasts were abundant in the stroma surrounding disrupted prostate epithelial structure without the BM. TGF-ß1 secretion from BPH-1 cells was increased by co-culturing with human primary cultured prostate fibroblasts. TNC mRNA expression was increased in fibroblasts co-culturing with BPH-1 cells and was suppressed by treatment with a TGF-ß RI kinase inhibitor. Moreover, in the castration group, the percentage of p-Smad2-positive cells was significantly higher in the stroma surrounding disrupted prostate epithelial structure without the BM. Our results demonstrate that castration-induced stromal remodeling disrupted the reconstituted human prostate-like epithelial structure and induced the appearance of TNC-positive fibroblasts accompanied by activation of TGF-ß signaling. The alteration of prostate stromal structure may be responsible for loss of the BM and epithelial cell polarity.

SOX11-induced decrease in vimentin and an increase in prostate cancer cell migration attributed to cofilin activity.

SOX11 is a transcription factor in the SOX family of genes that regulate multiple cellular events by influencing the expression of key genes in developmental, physiological, and tumorigenic cells. To elucidate the role of SOX11 in prostate cancer cells, PC-3 prostate cancer cells were cloned (S6 and S9 cells) to highly express SOX11. We demonstrated that both S6 and S9 lose vimentin expression, acquiring epithelial marker proteins, which indicates the Epithelial state phenotype. S6 and S9 cells have cancer-promoting characteristics that include higher migratory properties compared with control cells. The mechanisms that are responsible for the enhanced migration are cofilin activity and keratin 18 expression. TCGA (The Cancer Genome Atlas) dataset analysis revealed that metastatic prostate cancer tumors tend to have more SOX11 gene amplification compared with primary tumors. These results suggest the tumor promotive role and epithelial protein induction of SOX11 in prostate cancer cell.

Cancer-related gene mutations and intratumoral genetic heterogeneity in human epidermal growth factor receptor 2 heterogeneous gastric cancer.

Human epidermal growth factor receptor 2 (HER2) protein overexpression is associated with HER2 gene amplification, a critical driver oncogenetic change in gastric cancer. HER2 heterogeneity in advanced gastric cancer is associated with a poor prognosis and affects the clinical efficacy of trastuzumab. However, the mechanisms of HER2 heterogeneity are not fully understood. Here, we examined whether HER2 heterogeneous gastric cancer exhibited intratumoral genetic heterogeneity in other cancer-related genes. Two cases of advanced gastric cancer with HER2 heterogeneity were selected, and samples of HER2-positive and HER2-negative areas in each case were analyzed using a cancer-associated multiple gene panel. In both cases, TP53 mutations were observed in both HER2-positive and HER2-negative areas, whereas many of the potential driver and passenger mutations differed between HER2-positive and HER2-negative areas. Overall, our findings demonstrated that HER2 heterogeneous gastric cancer exhibited intratumoral genetic heterogeneity in other cancer-related genes and that the molecular mechanisms could differ between HER2-positive and -negative areas.

Systemic Dermatitis Model Mice Exhibit Atrophy of Visceral Adipose Tissue and Increase Stromal Cells via Skin-Derived Inflammatory Cytokines.

: Adipose tissue (AT) is the largest endocrine organ, producing bioactive products called adipocytokines, which regulate several metabolic pathways, especially in inflammatory conditions. On the other hand, there is evidence that chronic inflammatory skin disease is closely associated with vascular sclerotic changes, cardiomegaly, and severe systemic amyloidosis in multiple organs. In psoriasis, a common chronic intractable inflammatory skin disease, several studies have shown that adipokine levels are associated with disease severity. Chronic skin disease is also associated with metabolic syndrome, including abnormal tissue remodeling; however, the mechanism is still unclear. We addressed this problem using keratin 14-specific caspase-1 overexpressing transgenic (KCASP1Tg) mice with severe erosive dermatitis from 8 weeks of age, followed by re-epithelization. The whole body and gonadal white AT (GWAT) weights were decreased. Each adipocyte was large in number, small in size and irregularly shaped; abundant inflammatory cells, including activated CD4+ or CD8+ T cells and toll-like receptor 4/CD11b-positive activated monocytes, infiltrated into the GWAT. We assumed that inflammatory cytokine production in skin lesions was the key factor for this lymphocyte/monocyte activation and AT dysregulation. We tested our hypothesis that the AT in a mouse dermatitis model shows an impaired thermogenesis ability due to systemic inflammation. After exposure to 4 °C, the mRNA expression of the thermogenic gene uncoupling protein 1 in adipocytes was elevated; however, the body temperature of the KCASP1Tg mice decreased rapidly, revealing an impaired thermogenesis ability of the AT due to atrophy. Tumor necrosis factor (TNF)-α, IL-1ß and interferon (INF)-γ levels were significantly increased in KCASP1Tg mouse ear skin lesions. To investigate the direct effects of these cytokines, BL/6 wild mice were administered intraperitoneal TNF-α, IL-1ß and INF-γ injections, which resulted in small adipocytes with abundant stromal cell infiltration, suggesting those cytokines have a synergistic effect on adipocytes. The systemic dermatitis model mice showed atrophy of AT and increased stromal cells. These findings were reproducible by the intraperitoneal administration of inflammatory cytokines whose production was increased in inflamed skin lesions.

Antifibrotic Agent Pirfenidone Suppresses Proliferation of Human Pancreatic Cancer Cells by Inducing G0/G1 Cell Cycle Arrest.

BACKGROUND: Pirfenidone (PFD), which is an antifibrotic agent used for treatment of idiopathic pulmonary fibrosis, induces G0/G1 cell cycle arrest in fibroblasts. We hypothesized that PFD-induced G0/G1 cell cycle arrest might be achieved in other types of cells, including cancer cells. Here we investigated the effects of PFD on the proliferation of pancreatic cancer cells (PCCs) in vitro. METHOD: Human skin fibroblasts ASF-4-1 cells and human prostate stromal cells (PrSC) were used as fibroblasts. PANC-1, MIA PaCa-2, and BxPC-3 cells were used as human PCCs. Cell cycle and apoptosis were analyzed using flow cytometer. RESULTS: First, we confirmed that PFD suppressed cell proliferation of ASF-4-1 cells and PrSC and induced G0/G1 cell cycle arrest. Under these experimental conditions, PFD also suppressed cell proliferation and induced G0/G1 cell cycle arrest in all PCCs. In PFD-treated PCCs, expression of p21 was increased but that of CDK2 was not clearly decreased. Of note, PFD did not induce significant apoptosis among PCCs. CONCLUSIONS: These results demonstrated that the antifibrotic agent PFD might have antiproliferative effects on PCCs by inducing G0/G1 cell cycle arrest. This suggests that PFD may target not only fibroblasts but also PCCs in the tumor microenvironment of pancreatic cancer.

Establishment of an in vivo simulating co-culture assay platform for genotoxicity of multi-walled carbon nanotubes.

Engineered nanomaterials (ENM) are now used in a wide variety of fields, and, thus, their safety should urgently be assessed and secured. It has been suggested that inflammatory responses via the phagocytosis of ENM by macrophages is a key mechanism for their genotoxicity. The present study was conducted to establish a mechanism-based assay to evaluate the genotoxicity of ENM under conditions simulating an in vivo situation, featuring a co-culture system of murine lung resident cells (GDL1) and immune cells (RAW264.7). GDL1 were cultured with or without RAW264.7, exposed to a multi-walled carbon nanotube (MWCNT), and then analyzed for mutagenicity and underlying mechanisms. Mutation frequencies induced in GDL1 by the MWCNT were significantly greater with the co-existence of RAW264.7 than in its absence. Mutation spectra observed in GDL1 co-cultured with RAW264.7 were different from those seen in GDL1 cultured alone, but similar to those observed in the lungs of mice exposed to the MWCNT in vivo. Inflammatory cytokines, such as IL-1ß and TNF-α, were produced from RAW264.7 cells treated with the MWCNT. The generation of reactive oxygen species and the formation of 8-oxodeoxyguanosine in GDL1 exposed to the MWCNT were greater in the co-culture conditions than in the single culture conditions. Based on these findings, it is indicated that inflammatory responses are involved in the genotoxicity of MWCNT, and that the presently established, novel in vitro assay featuring a co-culture system of tissue resident cells with immune cells is suitable to evaluate the genotoxicity of ENM.

Molecular Mechanism Responsible for Fibronectin-controlled Alterations in Matrix Stiffness in Advanced Chronic Liver Fibrogenesis.

Fibrosis is characterized by extracellular matrix (ECM) remodeling and stiffening. However, the functional contribution of tissue stiffening to noncancer pathogenesis remains largely unknown. Fibronectin (Fn) is an ECM glycoprotein substantially expressed during tissue repair. Here we show in advanced chronic liver fibrogenesis using a mouse model lacking Fn that, unexpectedly, Fn-null livers lead to more extensive liver cirrhosis, which is accompanied by increased liver matrix stiffness and deteriorated hepatic functions. Furthermore, Fn-null livers exhibit more myofibroblast phenotypes and accumulate highly disorganized/diffuse collagenous ECM networks composed of thinner and significantly increased number of collagen fibrils during advanced chronic liver damage. Mechanistically, mutant livers show elevated local TGF-ß activity and lysyl oxidase expressions. A significant amount of active lysyl oxidase is released in Fn-null hepatic stellate cells in response to TGF-ß1 through canonical and noncanonical Smad such as PI3 kinase-mediated pathways. TGF-ß1-induced collagen fibril stiffness in Fn-null hepatic stellate cells is significantly higher compared with wild-type cells. Inhibition of lysyl oxidase significantly reduces collagen fibril stiffness, and treatment of Fn recovers collagen fibril stiffness to wild-type levels. Thus, our findings indicate an indispensable role for Fn in chronic liver fibrosis/cirrhosis in negatively regulating TGF-ß bioavailability, which in turn modulates ECM remodeling and stiffening and consequently preserves adult organ functions. Furthermore, this regulatory mechanism by Fn could be translated for a potential therapeutic target in a broader variety of chronic fibrotic diseases.

Primordial oscillations in life: Direct observation of glycolytic oscillations in individual HeLa cervical cancer cells.

We report the first direct observation of glycolytic oscillations in HeLa cervical cancer cells, which we regard as primordial oscillations preserved in living cells. HeLa cells starved of glucose or both glucose and serum exhibited glycolytic oscillations in nicotinamide adenine dinucleotide (NADH), exhibiting asynchronous intercellular behaviors. Also found were spatially homogeneous and inhomogeneous intracellular NADH oscillations in the individual cells. Our results demonstrate that starved HeLa cells may be induced to exhibit glycolytic oscillations by either high-uptake of glucose or the enhancement of a glycolytic pathway (Crabtree effect or the Warburg effect), or both. Their asynchronous collective behaviors in the oscillations were probably due to a weak intercellular coupling. Elucidation of the relationship between the mechanism of glycolytic dynamics in cancer cells and their pathophysiological characteristics remains a challenge in future.

[A Case of Fibromuscular Dysplasia in a Patient with Various Main Trunk Dissections in the Head and Neck over a Short Period].

We report a case of subarachnoid hemorrhage(SAH)from an intracranial vertebral artery(VA)dissection in a patient with fibromuscular dysplasia(FMD)who presented with headache. A 54-year-old woman complained of spontaneous occipital headache. The dilatation of the left VA was detected on magnetic resonance angiography(MRA). She was diagnosed with left VA dissection(headache onset type). After sudden onset of headache on the second day of hospitalization, her consciousness level, as defined by the Japan Coma Scale, was 300. Computed tomography(CT)revealed SAH. Cerebral angiography showed the dilatation of the left intracranial VA and contrast material pooling, which was suspected to be a sign of dissection. We performed VA intravascular ligation by coil embolization. The postoperative course was good but postoperative MRA revealed arterial wall irregularities in both the extra cranial internal carotid artery and the right VA. Cerebral angiography showed the presence of the string-of-beads sign at these arteries. She was diagnosed with FMD. SAH might develop during the follow-up period in patients with VA dissection, even those in whom the initial symptom is headache. In addition, cases of FMD might also be complicated by various lesions of the main trunk of the cerebral artery.

Adipocyte-derived monocyte chemotactic protein-1 (MCP-1) promotes prostate cancer progression through the induction of MMP-2 activity.

BACKGROUND: Obesity is known to be associated with prostate cancer development and progression, but the detailed mechanism is not clear. Monocyte chemotactic protein-1 (MCP-1) is secreted from cancer cells, stromal cells, and adipocytes, and it is involved in prostate cancer progression. Here we investigated the biological role of MCP-1 secreted from adipocytes for prostate cancer cells. METHODS: Human pre-adipocytes (HPAds) were cultured and differentiated to mature adipocytes. Conditioned medium (CM) from HPAd cells was obtained using phenol red-free RPMI1640 medium. We performed a cytokine membrane array analysis to detect cytokines in the CM. To characterize the physiological function of MCP-1 in the CM, we performed an MTT-assay, a wound-healing and invasion assay with anti-MCP-1 antibody using three prostate cancer cell lines: DU145, LNCaP, and PC-3. Matrix metalloproteinase (MMP)-2 and MMP-9 activities were evaluated by gelatin zymography. A qPCR and Western blotting were used to examine the mRNA and protein expression levels of MMP-2. RESULTS: The cytokine membrane array of the CM showed a strong signal of MCP-1compared to the control medium, and we thus focused our attention on MCP-1 in the CM. The CM up-regulated the cancer cell proliferation, and the neutralization by anti-MCP-1 antibody inhibited the proliferative effect of the prostate cancer cell lines. The CM greatly increased the invasive activity in the prostate cancer cell lines, and anti-MCP-1 antibody decreased the invasiveness. Gelatin zymography revealed that the CM markedly enhanced the enzymatic activity of MMP-2, and anti-MCP-1 antibody down-regulated its effect. MMP-2 mRNA expression was undetected and the MMP-2 protein level was unchanged between the control medium and CM in DU145 cells. CONCLUSIONS: MCP-1 from adipocytes enhances the growth and invasion activity of prostate cancer cells. The inhibition of MCP-1 derived from adipocytes might be an effective treatment for prostate cancer.

Comprehensive DNA adduct analysis reveals pulmonary inflammatory response contributes to genotoxic action of magnetite nanoparticles.

Nanosized-magnetite (MGT) is widely utilized in medicinal and industrial fields; however, its toxicological properties are not well documented. In our previous report, MGT showed genotoxicity in both in vitro and in vivo assay systems, and it was suggested that inflammatory responses exist behind the genotoxicity. To further clarify mechanisms underlying the genotoxicity, a comprehensive DNA adduct (DNA adductome) analysis was conducted using DNA samples derived from the lungs of mice exposed to MGT. In total, 30 and 42 types of DNA adducts were detected in the vehicle control and MGT-treated groups, respectively. Principal component analysis (PCA) against a subset of DNA adducts was applied and several adducts, which are deduced to be formed by inflammation or oxidative stress, as the case of etheno-deoxycytidine (εdC), revealed higher contributions to MGT exposure. By quantitative-LC-MS/MS analysis, εdC levels were significantly higher in MGT-treated mice than those of the vehicle control. Taken together with our previous data, it is suggested that inflammatory responses might be involved in the genotoxicity induced by MGT in the lungs of mice.