ABSTRACT
Tumors induce angiogenesis to acquire oxygen and nutrition from their adjacent microenvironment. Tumor angiogenesis has been believed to be induced primarily by the secretion of vascular endothelial growth factor-A (VEGF-A) from various tumors. VEGF-A binds to VEGF receptor 2 (VEGFR2), resulting in subsequent activation of cellular substances regulating cell proliferation, survival, and angiogenesis. Antiangiogenic therapies targeting the VEGF-A/VEGFR2 axis, including bevacizumab and ramucirumab, humanized monoclonal antibodies against VEGF-A and VEGFR2, respectively, have been proposed as a promising strategy aimed at preventing tumor growth, invasion, and metastasis. Phase III clinical trials using bevacizumab and ramucirumab have shown that not all tumor patients benefit from such antiangiogenic agents, and that some patients who initially benefit subsequently become less responsive to these antibodies, suggesting the possible existence of VEGF-independent angiogenic factors. In this review, we focus on VEGF-independent and VEGFR2-dependent tumor angiogenesis, as well as VEGFR2-independent tumor angiogenesis. Additionally, we discuss VEGF-independent angiogenic factors which have been reported in previous studies. Various molecular targeting drugs are currently being evaluated as potential antitumor therapies. We expect that precision medicine will permit the development of innovative antiangiogenic therapies targeting individual angiogenic factors selected on the basis of the genetic screening of tumors.
Subject(s)
Neoplasms , Vascular Endothelial Growth Factor A , Angiogenesis Inducing Agents , Angiogenesis Inhibitors/therapeutic use , Bevacizumab/pharmacology , Bevacizumab/therapeutic use , Humans , Neoplasms/drug therapy , Neovascularization, Pathologic/drug therapy , Neovascularization, Pathologic/metabolism , Tumor Microenvironment , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor Receptor-2/metabolismABSTRACT
Platelets are a major source of microRNAs (miRNAs) in blood. Relationships between circulating platelet-derived miRNAs were investigated to elucidate their significance as biomarkers. Total miRNAs in serum were analyzed using the 3D-Gene miRNA Oligo chip. Among 22 miRNAs that are included in platelets and play functional roles, sufficient miRNA levels for comparison were detected for 11 miRNAs (let-7b-5p, miR-16-5p, miR-17-5p, miR-24-3p, miR-107, miR-126-3p, miR-150-3p, miR-191-5p, miR-197-3p, miR-223-3p, and miR-326). Among 55 pairs prepared by these miRNAs, relatively strong correlations (Spearman's correlation coefficient >0.8) were shown between miRNAs of 7 pairs including let-7b-5p and miR-16-5p, let-7b-5p and miR-17-5p, let-7b-5p and miR-107, miR-16-5p and miR-17-5p, miR-16-5p and miR-107, miR-17-5p and miR-107, and miR-107 and miR-126-3p. In principal component analysis, the first principal component consisted of let-7b-5p, miR-16-5p, miR-17-5p, miR-107, miR-126-3p, and miR-191-5p. These six miRNAs may be useful biomarkers that reflect platelet condition and function.
Subject(s)
Blood Platelets/metabolism , MicroRNAs/metabolism , Adult , Humans , Male , Middle AgedABSTRACT
Human leukocyte antigen (HLA)-haploidentical stem cell transplantation (haplo-SCT) is associated with a high incidence of cytomegalovirus (CMV) infection, probably originating from the delayed reconstitution of CMV-specific T cell immunity. There have been few reports on the presence of CMV-specific cytotoxic T lymphocytes (CMV-CTLs) after haplo-SCT. We have studied CMV-specific immune reconstitution by measuring the absolute number of CMV-CTLs using a flow cytometry method with HLA-A2-restricted NLVPMVATV peptide dextramers. We examined the association between reconstitution patterns of CMV-CTLs and the duration of CMV antigenemia in 15 patients who underwent first allogeneic SCT from HLA-haploidentical-related donors with HLA-A2. In seven and eight patients, CMV antigenemia consecutively resolved for more than 4 weeks (the CMV antigenemia 'resolved' group) and intermittently persisted (the CMV antigenemia 'persistent' group) during a 100-day observation period, respectively. The group of the seven patients, in whom levels of CMV antigenemia were reduced to zero, had a significantly lower maximum level of CMV antigenemia than the CMV antigenemia persistent group. In contrast, the CMV antigenemia persistent group had a significantly higher maximum level of CMV-CTLs, but the levels took longer to peak. Despite no difference in general lymphocyte recovery between the two groups, the CMV antigenemia resolved group had significantly higher median CMV-CTL counts than the CMV antigenemia persistent group at 6 weeks after onset of CMV infection. Flow cytometry analysis of CMV-CTLs is a convenient method of monitoring reconstitution of CMV-specific lymphocyte immunity following haplo-SCT.
Subject(s)
Cytomegalovirus Infections/diagnosis , Cytomegalovirus Infections/immunology , Cytomegalovirus/immunology , HLA Antigens/immunology , Hematopoietic Stem Cell Transplantation/adverse effects , T-Lymphocytes, Cytotoxic/immunology , Adult , Cytomegalovirus/isolation & purification , Cytomegalovirus Infections/mortality , Female , Follow-Up Studies , Haplotypes , Humans , Male , Middle Aged , Survival Rate/trends , T-Lymphocytes, Cytotoxic/virology , Transplantation, HomologousABSTRACT
Thrombomodulin (TM), a transmembrane glycoprotein on vascular endothelial cells, is a naturally occurring anticoagulant. Recombinant human soluble TM (rTM), composed of the extracellular domain of TM, also shows anti-coagulant and anti-inflammatory activity, but the effects of rTM on microangiopathy remain unclear. We reported that FK506 induced endothelial dysfunction through inactivation of Akt and extracellular-regulated kinase 1/2 using a three-dimensional culture blood vessel model. In the present study, we examined the effects of rTM on FK506-induced endothelial dysfunction. We found that rTM suppressed FK506-induced endothelial cell death, but not the breakdown of capillary-like tube structures. rTM prevented FK506-induced inactivation of Akt, but not of extracellular-regulated kinase 1/2. Akt inhibition by LY294002 abrogated the preventive effect of rTM on FK506-induced Akt inactivation and the suppressive effect of rTM on FK506-induced cell death. These results suggest that rTM attenuates FK506-induced endothelial dysfunction through prevention of Akt inactivation.
Subject(s)
Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Recombinant Proteins/pharmacology , Tacrolimus/pharmacology , Thrombomodulin , Cell Death/drug effects , Cell Line , Chromones/pharmacology , Enzyme Inhibitors/pharmacology , Fibroblast Growth Factor 2/metabolism , HMGB1 Protein/metabolism , Humans , Immunosuppressive Agents/pharmacology , Morpholines/pharmacology , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Vascular Endothelial Growth Factor A/metabolismABSTRACT
BACKGROUND: Diffuse malignant peritoneal mesothelioma (DMPM) is an aggressive malignant tumor of mesothelial origin that shows a limited response to cytoreductive surgery along with intraperitoneal chemotherapy. Therefore, diagnosing DMPM early is very important. Reactive oxygen species play an important role in asbestos toxicity, which is associated with the pathogenesis of DMPM growth. Thioredoxin-1 (TRX) is a small redox-active protein that demonstrates antioxidative activity associated with tumor growth. Here, we investigated the serum levels of TRX in patients with DMPM and compared them with those of a population that had been exposed to asbestos but did not have DMPM. STUDY: The serum concentrations of TRX were measured in 15 DMPM patients and 34 individuals with benign asbestos-related diseases. RESULTS: We demonstrated that the patients with DMPM had significantly higher serum levels of TRX than the population that had been exposed to asbestos but did not have DMPM. CONCLUSIONS: Our data suggest that serum TRX concentration is a useful serum marker for DMPM.
Subject(s)
Biomarkers, Tumor/blood , Mesothelioma/diagnosis , Peritoneal Neoplasms/blood , Peritoneal Neoplasms/diagnosis , Thioredoxins/blood , Adult , Aged , Aged, 80 and over , Early Detection of Cancer , Female , Humans , In Vitro Techniques , Male , Mesothelioma/blood , Middle Aged , Predictive Value of Tests , ROC Curve , Sampling Studies , Sensitivity and SpecificityABSTRACT
A phenotype switch from contractile type to proliferative type of arterial smooth muscle cells is known as dedifferentiation, but to the best of our knowledge, little is known about redifferentiation of coronary artery smooth muscle cells. The purpose of the present study was to determine in vitro culture conditions for inducing redifferentiation of coronary artery smooth muscle cells. In addition, the present study aimed to determine protein markers for detection of redifferentiated arterial smooth muscle cells. Human coronary artery smooth muscle cells (HCASMCs) were cultured in the presence or absence of growth factors, including epidermal growth factor, fibroblast growth factor-B and insulin. Protein expression and migration activity of HCASMCs were evaluated using western blotting and migration assay, respectively. In HCASMCs 5 days after 100% confluency, expression levels of α-smooth muscle actin (α-SMA), calponin, caldesmon and SM22α were significantly increased, while expression levels of proliferation cell nuclear antigen (PCNA) and S100A4 and migration activity were significantly decreased, compared with the corresponding levels just after reaching 100% confluency, indicating that redifferentiation occurred. Redifferentiation was also induced in a low-density culture of HCASMCs in the medium without growth factors. When the culture medium for confluent cells was replaced daily with fresh medium, the expression levels of α-SMA, caldesmon, SM22α, PCNA and S100A4 and migration activity were not significantly different but the calponin expression was significantly increased compared with the levels in dedifferentiated cells just after reaching 100% confluency. Thus, redifferentiation was induced in HCASMCs by deprivation of growth factors from culture medium. The results suggested that α-SMA, caldesmon and SM22α, but not calponin, are markers of redifferentiation of HCASMCs.
ABSTRACT
BACKGROUND: Skeletal muscle comprises almost 40% of the human body and is essential for movement, structural support and metabolic homeostasis. Size of multinuclear skeletal muscle is stably maintained under steady conditions with the sporadic fusion of newly produced myocytes to compensate for the muscular turnover caused by daily wear and tear. It is becoming clear that microvascular pericytes (PCs) exhibit myogenic activity. However, whether PCs act as myogenic stem cells for the homeostatic maintenance of skeletal muscles during adulthood remains uncertain. METHODS: We utilized PC-fused myofibers using PC-specific lineage tracing mouse (NG2-CreERT/Rosa-tdTomato) to observe whether muscle resident PCs have myogenic potential during daily life. Genetic PC deletion mouse model (NG2-CreERT/DTA) was used to test whether PC differentiates to myofibers for maintenance of muscle structure and function under homeostatic condition. RESULTS: Under steady breeding conditions, tdTomato-expressing PCs were infused into myofibers, and subsequently, PC-derived nuclei were incorporated into myofibers. Especially in type-I slow-type myofibers such as the soleus, tdTomato+ myofibers were already observed 3 days after PC labeling; their ratio reached a peak (approximately 80%) within 1 month and was maintained for more than 1 year. Consistently, the NG2+ PC-specific deletion induced muscular atrophy in a slow-type myofiber-specific manner under steady breeding conditions. The number of myonucleus per volume of each myofiber was constant during observation period. CONCLUSIONS: These findings demonstrate that the turnover of myonuclei in slow-type myofibers is relatively fast, with PCs acting as myogenic stem cells-the suppliers of new myonuclei under steady conditions-and play a vital role in the homeostatic maintenance of slow-type muscles.
Subject(s)
Muscle, Skeletal , Pericytes , Animals , Humans , Mice , Cell Nucleus , Homeostasis , Muscular AtrophyABSTRACT
Malignant mesothelioma is an aggressive tumor arising from mesothelial cells of serous membranes. Src family kinases (SFKs) have a pivotal role in cell adhesion, proliferation, survival and apoptosis. Here, we examined the effect of SFK inhibitors in NCI-H2052, ACC-MESO-4 and NCI-H28 cells, mesothelioma cell lines and Met5A, a human non-malignant mesothelial cell line. We found that PP2, a selective SFK inhibitor, inhibited SFK activity and induced apoptosis mediated by caspase-8 in NCI-H28 but not Met5A, NCI-H2052 and ACC-MESO-4 cells. Src, Yes, Fyn and Lyn protein, which are members of the SFK, were expressed in these cell lines, whereas NCI-H28 cells were deficient in Fyn protein. Small interfering RNA (siRNA) targeting Fyn facilitated PP2-induced apoptosis mediated by caspase-8 in NCI-H2052 and ACC-MESO-4 cells. PP2 reduced Lyn protein levels and suppressed SFK activity in all mesothelioma cell lines. Lyn siRNA induced caspase-8 activation and apoptosis in NCI-H28 cells but not in NCI-H2052 and ACC-MESO-4 cells. However, double RNA interference knockdown of Fyn and Lyn induced apoptosis accompanied by caspase-8 activation in NCI-H2052 and ACC-MESO-4 cells. Dasatinib, an inhibitor of multi-tyrosine kinases including SFK, also inhibited SFK activity and induced reduction of Lyn protein levels, caspase-8 activation and apoptosis in NCI-H28 cells but not in other cell lines. Present study suggests that SFK inhibitors induce caspase-8-dependent apoptosis caused by reduction of Lyn protein in Fyn-deficient mesothelioma cells.
Subject(s)
Apoptosis/physiology , Mesothelioma/metabolism , Mesothelioma/pathology , Proto-Oncogene Proteins c-fyn/deficiency , src-Family Kinases/antagonists & inhibitors , src-Family Kinases/metabolism , Apoptosis/drug effects , Caspase 8/genetics , Caspase 8/metabolism , Cell Line, Tumor , Humans , Mesothelioma/genetics , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-fyn/genetics , Proto-Oncogene Proteins c-fyn/metabolism , Proto-Oncogene Proteins c-yes/genetics , Proto-Oncogene Proteins c-yes/metabolism , Pyrimidines/pharmacology , RNA, Messenger/genetics , RNA, Small Interfering/genetics , Signal Transduction/drug effects , Signal Transduction/genetics , Transcription, Genetic/drug effects , src-Family Kinases/geneticsABSTRACT
Mismatched human leukocyte antigens (HLAs) on leukemic cells can be targeted by donor T cells in HLA-mismatched/haploidentical stem cell transplantation. In two cases of acute myeloid leukemia with t(6;11)(q27;q23) abnormality presented here, flow cytometry analysis showed a lack of HLA-A unshared between recipients and donors in relapsing leukemic cells after HLA-haploidentical transplantation. However, high-resolution HLA genotyping showed that one case lacked a corresponding HLA haplotype, whereas the other preserved it. These cases suggest that leukemic cells, which lacked mismatched HLA expression, might have an advantage in selective expansion under donor T-cell immune surveillance after HLA-haploidentical transplantation. Most importantly, down-regulation of unshared HLA expression potentially occurs by genetic alterations other than loss of HLA alleles.
Subject(s)
Bone Marrow Transplantation , HLA Antigens/genetics , Hematopoietic Stem Cell Transplantation , Leukemia, Myeloid, Acute/genetics , Adult , Chromosomes, Human, Pair 11/immunology , Chromosomes, Human, Pair 6/immunology , Female , Graft vs Host Disease/genetics , HLA Antigens/immunology , Haplotypes , Histocompatibility , Histocompatibility Testing , Humans , Leukemia, Myeloid, Acute/immunology , Recurrence , T-Lymphocytes/immunology , Tissue Donors , Translocation, Genetic/immunology , Transplantation, HomologousABSTRACT
The formation of mature vasculature through angiogenesis is essential for adequate wound healing, such that blood-borne cells, nutrients, and oxygen can be delivered to the remodeling skin area. Neovessel maturation is highly dependent on the coordinated functions of vascular endothelial cells and perivascular cells, namely pericytes (PCs). However, the underlying mechanism for vascular maturation has not been completely elucidated, and its role in wound healing remains unclear. In this study, we investigated the role of Ninjurin-1 (Ninj1), a new molecule mediating vascular maturation, in wound healing using an inducible PC-specific Ninj1 deletion mouse model. Ninj1 expression increased temporarily in NG2-positive PCs in response to skin injury. When tamoxifen treatment induced a decreased Ninj1 expression in PCs, the neovessels in the regenerating wound margins were structurally and functionally immature, but the total number of microvessels was unaltered. This phenotypic change is associated with a reduction in PC-associated microvessels. Wound healing was significantly delayed in the NG2-specific Ninj1 deletion mouse model. Finally, we showed that Ninj1 is a crucial molecule that mediates vascular maturation in injured skin tissue through the interaction of vascular endothelial cells and PCs, thereby inducing adequate and prompt wound healing.
ABSTRACT
Malignant mesothelioma is an aggressive tumor of serosal surfaces, which is refractory to current treatment options. Arsenic trioxide (As2O3) is used clinically to treat acute promyelocytic leukemia, and also to inhibit proliferation of several solid tumors including hepatoma, esophageal, and gastric cancer in vitro. Here we found that As2O3 inhibited cell viability of a mesothelioma cell line, NCI-H2052. As2O3 induced apoptosis of NCI-H2052 cells, which was accompanied by activation of c-Jun NH2-terminal kinase (JNK)1/2, extracellular signal-regulated kinase (ERK)1/2, and caspase-3. zVAD-fmk, a broad-spectrum caspase inhibitor, inhibited As2O3-induced apoptosis and activation of caspase-3, but not that of JNK1/2 and ERK1/2. Small interfering RNAs (siRNAs) targeting JNK1/2 suppressed As2O3-induced caspase-3 activation and apoptosis, indicating that JNK1/2 regulate As2O3-induced apoptosis though caspase cascade. Furthermore, JNK1 siRNA abrogated As2O3-induced JNK2 phosphorylation and JNK2 siRNA abrogated As2O3-induced JNK1 phosphorylation, suggesting that JNK1 and JNK2 interact with each other. Moreover, JNK1 siRNA, but not JNK2 siRNA, abrogated As2O3-induced ERK1/2 phosphorylation. JNK2 siRNA together with PD98059, a specific MAPK/ERK kinase inhibitor, suppressed As2O3-induced apoptosis more significantly than JNK2 siRNA alone. These results indicated that As2O3 induces apoptosis of NCI-H2052 cells mainly through JNK1/2 activation, and that ERK1/2 is involved in As2O3-induced apoptosis when JNK1/2 are inactivated.
Subject(s)
Apoptosis/drug effects , Arsenicals/pharmacology , Extracellular Signal-Regulated MAP Kinases/metabolism , JNK Mitogen-Activated Protein Kinases/metabolism , Mesothelioma/enzymology , Mesothelioma/pathology , Oxides/pharmacology , Arsenic Trioxide , Caspases/metabolism , Cell Line, Tumor , Drug Screening Assays, Antitumor , Enzyme Activation/drug effects , Humans , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Mitogen-Activated Protein Kinase 8/metabolism , Mitogen-Activated Protein Kinase 9/metabolismABSTRACT
We recently reported that propolis suppresses tumor-induced angiogenesis through tube formation inhibition and apoptosis induction in endothelial cells. However, molecular mechanisms underlying such angiogenesis suppression by propolis have not been fully elucidated. The aim of this study was to investigate the effects of ethanol extract of Brazilian propolis (EEBP) on two major survival signals, extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt, and to elucidate whether changes in these signals were actually involved in antiangiogenic effects of the propolis. Detection by western blotting revealed that EEBP suppressed phosphorylation of ERK1/2, but not that of Akt. Pharmacological inhibition by U0126 demonstrated that ERK1/2 inactivation alone was enough to inhibit tube formation and induce apoptosis. It was also shown that EEBP and U0126 similarly induced activation of caspase-3 and cleavage of poly ADP-ribose polymerase (PARP) and lamin A/C, all of which are molecular markers of apoptosis. These results indicate that inhibition of survival signal ERK1/2, and subsequent induction of apoptosis, is a critical mechanism of angiogenesis suppression by EEBP.
ABSTRACT
We recently reported that a broad-spectrum caspase inhibitor zVAD-fmk failed, while p38 inhibitor SB203580 succeeded, to prevent chromatin condensation and nuclear fragmentation induced by hypoxia in tube-forming HUVECs. In this study, we investigated the reasons for zVAD-fmk's inability to inhibit these morphological changes at the molecular level. The inhibitor effectively inhibited DNA ladder formation and activation of caspase-3 and -6, but it surprisingly failed to inhibit caspase-7 activation. On the other hand, SB203580 successfully inhibited all of these molecular events. When zLEHD-fmk, which specifically inhibits initiator caspase-9 upstream of caspase-3, was used, it inhibited caspase-3 activation but failed to inhibit caspase-6 and -7 activation. It also failed to inhibit hypoxia-induced chromatin condensation, nuclear fragmentation and DNA ladder formation. Taken together, our results indicate that, during hypoxia, caspase-7 is responsible for chromatin condensation and nuclear fragmentation while caspase-6 is responsible for DNA ladder formation.
Subject(s)
Apoptosis/physiology , Caspase 3/metabolism , Caspase 6/metabolism , Caspase 7/metabolism , Endothelial Cells/metabolism , Actins/metabolism , Amino Acid Chloromethyl Ketones/pharmacology , Apoptosis/drug effects , Caspase 9/metabolism , Caspase Inhibitors , Cell Hypoxia/physiology , Cell Line , Cell Nucleus/drug effects , Chromatin/drug effects , Cysteine Proteinase Inhibitors/pharmacology , DNA Breaks/drug effects , DNA Breaks, Double-Stranded/drug effects , Endothelial Cells/cytology , Endothelial Cells/drug effects , Humans , Imidazoles/pharmacology , Lamin Type A/metabolism , Models, Biological , Neovascularization, Physiologic/drug effects , Oligopeptides/pharmacology , Poly(ADP-ribose) Polymerases/metabolism , Protein Kinase Inhibitors/pharmacology , Pyridines/pharmacologyABSTRACT
Nonsmall cell lung cancer (NSCLC) accounts for over 80% of all diagnosed lung cancer cases. Lung cancer is the leading cause of cancerrelated deaths worldwide. Most NSCLC cells overexpress vascular endothelial growth factorA (VEGFA) which plays a pivotal role in tumour angiogenesis. Antiangiogenic therapies including VEGFA neutralisation have significantly improved the response rates, progressionfree survival and overall survival of patients with NSCLC. However, the median survival of these patients is shorter than 18 months, suggesting that NSCLC cells secrete VEGFindependent angiogenic factors, which remain unknown. We aimed to explore these factors in human NSCLC cell lines, A549, Lu99 and EBC1 using serumfree culture, to which only EBC1 cells could adapt. By mass spectrometry, we identified 1,007 proteins in the culture supernatant derived from EBC1 cells. Among the identified proteins, interleukin8 (IL8), macrophage migration inhibitory factor (MIF), galectin1, midkine (MK), IL18, galectin3, VEGFA, hepatomaderived growth factor (HDGF), osteopontin (OPN), connective tissue growth factor (CTGF) and granulin (GRN) are known to be involved in angiogenesis. Tube formation, neutralisation and RNA interference assays revealed that VEGFA and HDGF function as angiogenic factors in EBC1 cells. To confirm whether VEGFA and HDGF also regulate angiogenesis in the other NSCLC cell lines, we established a novel culture method. NSCLC cells were embedded in collagen gel and cultured threedimensionally. Tube formation, neutralisation and RNA interference assays using the threedimensional (3D) culture supernatant showed that VEGFA and HDGF were not angiogenic factors in Lu99 cells. By gene microarray in EBC1 and Lu99 cells, we identified 61 mRNAs expressed only in Lu99 cells. Among these mRNAs, brainderived neurotrophic factor (BDNF), fibroblast growth factor2 (FGF2) and FGF5 are known to be involved in angiogenesis. Tube formation and neutralisation assays clarified that FGF2 functions as an angiogenic factor in Lu99 cells. These results indicate that HDGF enhances VEGFdependent angiogenesis and that FGF2 is a VEGFindependent angiogenic factor in human NSCLC cells.
Subject(s)
Carcinoma, Non-Small-Cell Lung/metabolism , Culture Media, Serum-Free/analysis , Fibroblast Growth Factor 2/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Lung Neoplasms/metabolism , Vascular Endothelial Growth Factor A/metabolism , A549 Cells , Carcinoma, Non-Small-Cell Lung/genetics , Cell Culture Techniques , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Fibroblast Growth Factor 2/genetics , Gene Expression Regulation, Neoplastic , Humans , Intercellular Signaling Peptides and Proteins/genetics , Lung Neoplasms/genetics , Mass Spectrometry , Vascular Endothelial Growth Factor A/geneticsABSTRACT
BACKGROUND: Circulating microRNAs (miRNAs) have recently been proposed to be biomarkers for various diseases including cancer and cardiovascular disease. Erythrocytes are a major source of miRNAs in blood. However, it remains unknown how miRNA levels in serum are influenced by miRNAs in erythrocytes. In this study, we investigated the relationships among serum levels of miRNAs that are contained in erythrocytes. METHODS: Participants were middle-aged healthy Japanese men. Total miRNAs in serum from each participant were analyzed using the 3D-Gene miRNA Oligo chip. Relationships among the levels of eleven miRNAs (miR-103a-3p, -144-3p, -15a-5p, -16--5p, -26a-5p, -423-5p, -451a, -484, -486-5p, -92a-3p, and -93-5p) that have been reported to exist in erythrocytes were investigated by using correlation analysis. RESULTS: Among 55 pairs prepared by the above 11 miRNAs, there were significant correlations between miRNA levels of 31 pairs. In principal component analysis, 4 major erythrocyte-derived miRNAs, miR-16-5p, -451a, -486-5p and -92a-3p, were included in the first principal component. There were strong correlations between miR-16-5p and -451a levels (Spearman's rank correlation coefficient: 0.920) and between miR-486-5p and -92a-3p levels (Spearman's rank correlation coefficient: 0.863). CONCLUSION: There are significant associations among serum levels of erythrocyte-derived miRNAs, and these associations should be taken into account when considering the miRNAs as disease biomarkers.
Subject(s)
Erythrocytes/metabolism , Healthy Volunteers , MicroRNAs/blood , Female , Humans , Male , Middle AgedABSTRACT
Mortality from ischemic heart disease (IHD) is significantly lower in Japan than in Western countries. The purpose of this study was to investigate differences in circulating microRNA (miRNA) levels related to IHD in Austrians and Japanese. Participants were middle-aged healthy male Austrians (n = 20) and Japanese (n = 20). Total miRNAs in serum from each participant were analyzed using the 3D-Gene miRNA Oligo chip. Twenty-one miRNAs, previously reported as associated with IHD, were compared between Austrians and Japanese. The expression levels of miR-106a-5p, miR-135a-3p, miR-150-3p, miR-16-5p, miR-17-5p. miR-191-5p, miR-320b, miR-451a, miR-486-5p, miR-663b, and miR-92a-3p were significantly higher, while the miR-2861 expression level was significantly lower in Austrians as compared to Japanese. Both in Austrians and Japanese, there were significant positive correlations between serum expression levels of each pair of the above miRNAs except for miR-2861. The expression level of miR-2861 showed significant positive correlations with the expression levels of miR-106a-5p, miR-150-3p, miR-17-5p, miR-486-5p, miR-663b and miR-92a-3p in Austrians but not in Japanese. In pathway analysis, proinflammatory cytokine production in foam cells and collagen synthesis in vascular smooth muscle cells were associated with differentially expressed miRNAs. Difference in miRNA levels may contribute to lower cardiovascular risk in Japan than in Western countries.
Subject(s)
Biomarkers, Tumor/genetics , Circulating MicroRNA/genetics , Myocardial Ischemia/diagnosis , Austria/epidemiology , Biomarkers, Tumor/analysis , Case-Control Studies , Circulating MicroRNA/analysis , Gene Expression Profiling , Humans , Japan/epidemiology , Male , Middle Aged , Myocardial Ischemia/blood , Myocardial Ischemia/epidemiology , Myocardial Ischemia/genetics , Pilot Projects , PrognosisABSTRACT
A mouse jejunum, when incubated in vitro in Ussing chambers, was found to exhibit morphological deterioration of the villi with denudation of the epithelia (J Nutr Sci Vitaminol, 51: 406, 2005). Our study examined the involvement of apoptosis in an intestinal injury model by a DNA ladder assay and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. Electrophoresis of mucosal DNA revealed ladders, indicating the occurrence of DNA fragmentation. Cells with TUNEL-positive nuclei were detected among the villus epithelial cells (enterocytes), whereas they are rarely seen among crypt epithelial cells. These features were evident within 1 h after the start of incubation. Apoptotic death of the enterocytes was thus involved in the destruction of villi when incubated in Ussing chambers.
Subject(s)
Apoptosis , DNA Fragmentation , Enterocytes/pathology , Intestinal Mucosa/pathology , Jejunum/pathology , Animals , Animals, Outbred Strains , Diffusion Chambers, Culture , In Situ Nick-End Labeling , In Vitro Techniques , Male , MiceABSTRACT
We recently reported that hypoxia could induce the breakdown of capillary-like tubes formed by human umbilical vein endothelial cells (HUVECs) and that this breakdown was regulated by p38 and not by a caspase cascade, although the exact molecular mechanisms remain unknown. The aim of this study was to identify proteins that regulated hypoxia-induced tube breakdown through p38-regulated and caspase-independent mechanisms. The involvement of adhesion proteins, integrins, VE-cadherin, PECAM-1, and occludin was first investigated. Although some of these proteins decreased after hypoxia, none of them met the conditions of being quantitatively restored by p38 inhibition but not by caspase inhibition. We then conducted 2-D DIGE coupled with MALDI-TOF/TOF-MS to identify altered protein expression. The differential proteomic analysis of tube-forming HUVECs treated with normoxia or hypoxia and treated with hypoxia in the presence or absence of SB203580, a specific p38 inhibitor, revealed the involvement of heat shock proteins in this tube breakdown. We also confirmed that the amount of HSP27 and HSP70 changed in a p38-regulated and caspase-independent manner during hypoxia. Knocking down HSP27 expression using RNAi further augmented hypoxia-induced tube breakdown. Taken together, it was shown that p38-regulated and caspase-independent reduction of HSP27 plays an important role in hypoxia-induced tube breakdown.
Subject(s)
Endothelial Cells/pathology , Heat-Shock Proteins/metabolism , Hypoxia/pathology , Models, Biological , Neoplasm Proteins/metabolism , Proteomics , Umbilical Veins/cytology , p38 Mitogen-Activated Protein Kinases/physiology , Capillaries/enzymology , Capillaries/pathology , Cells, Cultured , Endothelial Cells/enzymology , HSP27 Heat-Shock Proteins , HSP70 Heat-Shock Proteins/physiology , Heat-Shock Proteins/antagonists & inhibitors , Heat-Shock Proteins/deficiency , Heat-Shock Proteins/genetics , Humans , Hypoxia/enzymology , Hypoxia/physiopathology , Molecular Chaperones , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/deficiency , Neoplasm Proteins/genetics , Proteome/metabolism , Umbilical Veins/enzymology , Umbilical Veins/pathologyABSTRACT
We recently reported that hypoxia induces chromatin condensation and cell nuclear fragmentation, morphological markers of apoptosis, to tube-forming HUVECs in an in vitro blood vessel model by activating p38 MAPK. In this report, we further examined what role p38 plays and how it is activated during hypoxia-induced apoptosis. First, in order to confirm that p38 can indeed induce apoptosis, the cells were treated with anisomycin, a p38 activator, during normoxia. The activator treatment induced apoptosis and activation of p38 and caspase-3 in a very short time, which indicated that p38 activation alone was sufficient to trigger apoptosis in tube-forming HUVECs. We then observed hypoxia-induced changes in intracellular signals, ERK1/2 and Akt. ERK1/2 inactivation was shown to occur prior to p38 activation and caspase-3 cleavage during hypoxia. On the other hand, anisomycin had no inhibitory effect on ERK1/2 activation during normoxia. It was also shown that the amount of Akt protein slightly decreased by either hypoxia or anisomycin treatment. We then investigated how these two survival signals, ERK1/2 and Akt, are involved in p38 activation by using MEK inhibitor U0126 and PI3K inhibitor LY294002. When tube-forming HUVECs were treated with U0126 or LY294002 during normoxia, the two inhibitors were able to induce apoptosis and activation of p38 and caspase-3 in a relatively short time. U0126 was able to inhibit ERK1/2 activation, but had almost no effect on Akt activation. In contrast, LY294002 was able to inhibit Akt activation, but had very little effect on ERK1/2 activation. These results indicate that ERK1/2 inactivation, rather than Akt decrease, is responsible for hypoxia-induced p38 activation. Taken together, our results strongly suggest that hypoxia-induced apoptosis is regulated through signal transduction in which inactivation of ERK1/2 leads to activation of p38, which then triggers caspase cascade as an execution mechanism of apoptosis.
Subject(s)
Apoptosis , Endothelial Cells/enzymology , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Neovascularization, Physiologic , Umbilical Veins/cytology , p38 Mitogen-Activated Protein Kinases/metabolism , Capillaries/enzymology , Caspase 3/metabolism , Cell Hypoxia , Cell Survival , Cells, Cultured , Endothelial Cells/cytology , Enzyme Activation , Humans , Models, Biological , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction , Umbilical Veins/enzymologyABSTRACT
A 56-year-old man was admitted to our hospital in July 2000 because of epigastralgia and back pain with past history of repeated upper abdominal pain due to acute pancreatitis since 1995. Abdominal computed tomography on admission showed a swelling in the pancreas head and several large pancreatic pseudocysts. He was diagnosed as acute pancreatitis based on abdominal pain, elevated pancreatic enzymes and computed tomography finding, and given 50 microg octreotide subcutaneously for the treatment of pancreatic pseudocysts. Within 3 hours after octreotide injection, he complained of upper abdominal pain and had an elevated serum amylase level. Abdominal pain disappeared after cessation of octreotide injection and the patient was discharged free from abdominal pain. Octreotide might cause acute pancreatitis by inducing spasm of the sphincter of Oddi. Careful check-up of the patients might be needed during treatment with octreotide.