RESUMO
Chronic hypoxia in utero causes intrauterine growth restriction (IUGR) of the fetus. IUGR infants are known to be at higher risk for neurodevelopmental disorders, but the mechanism is unclear. In this study, we analyzed the structure of the cerebral cortex using IUGR model rats generated through a reduced uterine perfusion pressure operation. IUGR rats exhibited thinner cerebral white matter and enlarged lateral ventricles compared with control rats. Expression of neuron cell markers, Satb2, microtubule-associated protein (MAP)-2, α-tubulin, and nestin was reduced in IUGR rats, indicating that neurons were diminished at various developmental stages in IUGR rats, from neural stem cells to mature neurons. However, there was no increase in apoptosis in IUGR rats. Cells positive for Ki67, a marker of cell proliferation, were reduced in neurons and all glial cells of IUGR rats. In primary neuron cultures, axonal elongation was impaired under hypoxic culture conditions mimicking the intrauterine environment of IUGR infants. Thus, in IUGR rats, chronic hypoxia in utero suppresses the proliferation of neurons and glial cells as well as axonal elongation, resulting in cortical thinning and enlarged lateral ventricles. Thrombopoietin (TPO), a platelet growth factor, inhibited the decrease in neuron number and promoted axon elongation in primary neurons under hypoxic conditions. Intraperitoneal administration of TPO to IUGR rats resulted in increases in the number of NeuN-positive cells and the area coverage of Satb2. In conclusion, suppression of neuronal proliferation and axonal outgrowth in IUGR rats resulted in cortical thinning and enlargement of lateral ventricles. TPO administration might be a novel therapeutic strategy for treating brain dysmaturation in IUGR infants.
Assuntos
Proliferação de Células , Retardo do Crescimento Fetal , Crescimento Neuronal , Neurônios , Fármacos Neuroprotetores , Ratos Sprague-Dawley , Trombopoetina , Animais , Retardo do Crescimento Fetal/patologia , Ratos , Neurônios/efeitos dos fármacos , Neurônios/patologia , Neurônios/metabolismo , Feminino , Proliferação de Células/efeitos dos fármacos , Gravidez , Crescimento Neuronal/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Células Cultivadas , Animais Recém-Nascidos , Córtex Cerebral/patologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismoRESUMO
The tumor suppressor p53 prevents cancer development by regulating dozens of target genes with diverse biological functions. Although numerous p53 target genes have been identified to date, the dynamics and function of the regulatory network centered on p53 have not yet been fully elucidated. We herein identified inhibitor of DNA-binding/differentiation-3 (ID3) as a direct p53 target gene. p53 bound the distal promoter of ID3 and positively regulated its transcription. ID3 expression was significantly decreased in clinical lung cancer tissues, and was closely associated with overall survival outcomes in these patients. Functionally, ID3 deficiency promoted the metastatic ability of lung cancer cells through its effects on the transcriptional regulation of CDH1. Furthermore, the ectopic expression of ID3 in p53-knockdown cells restored E-cadherin expression. Collectively, the present results demonstrate that ID3 plays a tumor-suppressive role as a downstream effector of p53 and impedes lung cancer cell metastasis by regulating E-cadherin expression.
Assuntos
Neoplasias Pulmonares , Humanos , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Proteínas Inibidoras de Diferenciação/genética , Proteínas Inibidoras de Diferenciação/metabolismo , Neoplasias Pulmonares/patologia , Proteínas de Neoplasias/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Macrophages play a role in nephrolithiasis, offering the possibility of developing macrophage-mediated preventive therapies. To establish a system for screening drugs that could prevent the formation of kidney stones, we aimed to develop a model using human induced pluripotent stem cell (iPSC)-derived macrophages to study phagocytosis of calcium oxalate monohydrate (COM) crystals. Human iPSCs (201B7) were cultured. CD14+ monocytes were recovered using a stepwise process that involved the use of growth factors and cytokines. These cells were then allowed to differentiate into M1 and M2 macrophages. The macrophages were co-cultured with COM crystals and used in the phagocytosis experiments. Live cell imaging and polarized light observation via super-resolution microscopy were used to visualize phagocytosis. Localization of phagocytosed COM crystals was observed using transmission electron microscopy. Intracellular fluorescence intensity was measured using imaging cytometry to quantify phagocytosis. Human iPSCs successfully differentiated into M1 and M2 macrophages. M1 macrophages adhered to the culture plate and moved COM crystals from the periphery to cell center over time, whereas M2 macrophages did not adhere to the culture plate and actively phagocytosed the surrounding COM crystals. Fluorescence assessment over a 24-h period showed that M2 macrophages exhibited higher intracellular fluorescence intensity (5.65-times higher than that of M1 macrophages at 4.5 h) and maintained this advantage for 18 h. This study revealed that human iPSC-derived macrophages have the ability to phagocytose COM crystals, presenting a new approach for studying urinary stone formation and highlighting the potential of iPSC-derived macrophages as a tool to screen nephrolithiasis-related drugs.
Assuntos
Células-Tronco Pluripotentes Induzidas , Cálculos Renais , Humanos , Oxalato de Cálcio/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Macrófagos/metabolismo , Fagocitose , Cálculos Renais/metabolismoRESUMO
Cardiac glycosides (CGs) have been used for decades to treat heart failure and arrhythmic diseases. Recent non-clinical and epidemiological findings have suggested that CGs exhibit anti-tumor activities. Therefore, CGs may be repositioned as drugs for the treatment of cancer. A detailed understanding of the anti-cancer mechanisms of CGs is essential for their application to the treatment of targetable cancer types. To elucidate the factors associated with the anti-tumor effects of CGs, we performed transcriptome profiling on human multiple myeloma AMO1 cells treated with periplocin, one of the CGs. Periplocin significantly down-regulated the transcription of MYC (c-Myc), a well-established oncogene. Periplocin also suppressed c-Myc expression at the protein levels. This repression of c-Myc was also observed in several cell lines. To identify target proteins for the inhibition of c-Myc, we generated CG-resistant (C9) cells using a sustained treatment with digoxin. We confirmed that C9 cells acquired resistance to the inhibition of c-Myc expression and cell proliferation by CGs. Moreover, the sequencing of genomic DNA in C9 cells revealed the mutation of D128N in α1-Na/K-ATPase, indicating the target protein. These results suggest that CGs suppress c-Myc expression in cancer cells via α1-Na/K-ATPase, which provides further support for the anti-tumor activities of CGs.
Assuntos
Glicosídeos Cardíacos , Humanos , Glicosídeos Cardíacos/farmacologia , Linhagem Celular , Proliferação de Células , Perfilação da Expressão Gênica , Adenosina TrifosfatasesRESUMO
Therapeutic hypothermia (TH) provides neuroprotection. However, the cellular mechanisms underlying the neuroprotective effects of TH are not fully elucidated. Regulation of microglial activation has the potential to treat a variety of nervous system diseases. Transient receptor potential vanilloid 4 (TRPV4), a nonselective cation channel, is activated by temperature stimulus at 27-35 °C. Although it is speculated that TRPV4 is associated with the neuroprotective mechanisms of TH, the role of TRPV4 in the neuroprotective effects of TH is not well understood. In the present study, we investigated whether hypothermia attenuates microglial activation via TRPV4 channels. Cultured microglia were incubated under normothermic (37 °C) or hypothermic (33.5 °C) conditions following lipopolysaccharide (LPS) stimulation. Hypothermic conditions suppressed the expression of pro-inflammatory cytokines, inducible nitric oxide synthase, and the number of phagocytic microglia. AMP-activated protein kinase (AMPK)-NF-κB signaling was inhibited under hypothermic conditions. Furthermore, hypothermia reduced neuronal damage induced by LPS-treated microglial cells. Treatment with TRPV4 antagonist in normothermic culture replicated the suppressive effects of hypothermia on microglial activation and microglia-induced neuronal damage. In contrast, treatment with a TRPV4 agonist in hypothermic culture reversed the suppressive effect of hypothermia. These findings suggest that TH suppresses microglial activation and microglia-induced neuronal damage via the TRPV4-AMPK-NF-κB pathway. Although more validation is needed to consider differences according to age, sex, and specific central nervous system regions, our findings may offer a novel therapeutic approach to complement TH.
Assuntos
Antineoplásicos , Hipotermia , Fármacos Neuroprotetores , Humanos , NF-kappa B/metabolismo , Microglia/metabolismo , Canais de Cátion TRPV/metabolismo , Fármacos Neuroprotetores/farmacologia , Hipotermia/metabolismo , Lipopolissacarídeos/toxicidade , Proteínas Quinases Ativadas por AMP/metabolismo , Antineoplásicos/farmacologia , Óxido Nítrico/metabolismoRESUMO
Atherosclerosis is a persistent inflammatory state that contributes significantly to cardiovascular disease, a primary cause of mortality worldwide. Enhanced lipid uptake by macrophages and their transformation into foam cells play a key role in the development of atherosclerosis. Recent studies using in vivo mouse models indicated that activation of AMPK has anti-atherosclerotic effects by upregulating the expression of cholesterol efflux transporters in foam cells and promoting cholesterol efflux. However, the pathway downstream of AMPK that contributes to elevated expression of cholesterol efflux transporters remains unclear. In this study, we found that activation of AMPK by AICAR and metformin inhibits foam cell formation via suppression of mTOR in macrophages. Specifically, activation of AMPK indirectly reduced the phosphorylation level of mTOR at Ser2448 and promoted the expression of cholesterol efflux transporters and cholesterol efflux. These inhibitory effects on foam cell formation were counteracted by mTOR activators. Metformin, a more nonspecific AMPK activator than AICAR, appears to inhibit foam cell formation via anti-inflammatory effects in addition to suppression of the mTOR pathway. The results of this study suggest that the development of new drugs targeting AMPK activation and mTOR inhibition may lead to beneficial results in the prevention and treatment of atherosclerosis.
Assuntos
Aterosclerose , Metformina , Animais , Camundongos , Proteínas Quinases Ativadas por AMP/metabolismo , Macrófagos/metabolismo , Colesterol/metabolismo , Células Espumosas , Serina-Treonina Quinases TOR/metabolismo , Metformina/farmacologia , Metformina/metabolismo , Aterosclerose/metabolismo , Transportador 1 de Cassete de Ligação de ATP/metabolismoRESUMO
BACKGROUND: Neuroblastoma is one of the most common childhood solid tumors. Because tumor suppressor genes are often hypermethylated in cancers, DNA methylation has emerged as a target for cancer therapeutics. Nanaomycin A, an inhibitor of DNA methyltransferase 3B, which mediates de novo DNA methylation, reportedly induces death in several types of human cancer cells. OBJECTIVE: To study the antitumor activity of nanaomycin A against neuroblastoma cell lines and its mechanism. METHODS: The anti-tumor effect of nanaomycin A on neuroblastoma cell lines was evaluated based on cell viability, DNA methylation levels, apoptosis-related protein expression, and neuronal-associated mRNA expression. RESULTS: Nanaomycin A decreased genomic DNA methylation levels and induced apoptosis in human neuroblastoma cells. Nanaomycin A also upregulated the expression of mRNAs for several genes related to neuronal maturation. CONCLUSIONS: Nanaomycin A is an effective therapeutic candidate for treating neuroblastoma. Our findings also suggest that the inhibition of DNA methylation is a promising anti-tumor therapy strategy for neuroblastoma.
Assuntos
Naftoquinonas , Neuroblastoma , Humanos , Criança , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Neuroblastoma/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Naftoquinonas/farmacologia , Naftoquinonas/uso terapêutico , Metilação de DNA , Linhagem Celular Tumoral , DNA Metiltransferase 3BRESUMO
Genetic abnormalities induce the DNA damage response (DDR), which enables DNA repair at cell cycle checkpoints. Although the DDR is thought to function in preventing the onset and progression of cancer, DDR-related proteins are also thought to contribute to tumorigenesis, tumor progression, and drug resistance by preventing irreparable genomic abnormalities from inducing cell death. In the present study, the combination of ataxia telangiectasia-mutated serine/threonine kinase (ATM) and checkpoint kinase 1 (Chk1) inhibition exhibited synergistic antitumor effects and induced synergistic lethality in colorectal cancer cells at a low dose. The ATM and Chk1 inhibitors synergistically promoted the activation of cyclin-dependent kinase 1 by decreasing the phosphorylation levels of T14 and Y15. Furthermore, the combined treatment increased the number of sub-G1-stage cells, phospho-histone H2A.X-positive cells, and TdT-mediated dUTP nick-end labeling-positive cells among colon cancer cells, suggesting that the therapy induces apoptosis. Finally, the combined treatment exhibited a robust antitumor activity in syngeneic tumor model mice. These findings should contribute to the development of new treatments for colorectal cancer that directly exploit the genomic instability of cancer cells.
RESUMO
BACKGROUND: Small-for-gestational-age (SGA) infants are at increased risk for transient thrombocytopenia. The aim of this study was to determine whether thrombocytopenia in human SGA infants is due to insufficient thrombopoietin (TPO) production. METHODS: A prospective study of 202 infants with gestational age less than 37 weeks was conducted; 30 of them were SGA infants, and 172 were non-SGA infants. Thrombocytopenia was seen in 17 of 30 SGA infants and 40 of 172 non-SGA infants. RESULTS: Platelet counts were significantly lower in the SGA group than in the non-SGA group at the time of the lowest platelet count within 72 h of birth. The platelet count and immature platelet fraction (IPF) were negatively correlated in non-SGA infants, but not in SGA infants. In addition, the platelet count and TPO were negatively correlated in non-SGA infants. IPF and TPO were significantly lower in SGA than in non-SGA infants with thrombocytopenia. CONCLUSION: IPF increased with thrombocytopenia to promote platelet production in non-SGA infants due to increasing TPO, but not in SGA infants. This study found an association between insufficient TPO production and thrombocytopenia in SGA infants. In addition, this study is important for understanding the etiology of thrombocytopenia in SGA infants. IMPACT: The immature platelet fraction was low, and serum thrombopoietin was not increased in small-for-gestational-age (SGA) infants with thrombocytopenia. Thrombocytopenia in SGA infants is due to insufficient thrombopoietin production. This study is important for understanding the etiology of thrombocytopenia in SGA infants.
Assuntos
Trombocitopenia , Trombopoetina , Feminino , Humanos , Lactente , Estudos Prospectivos , Contagem de Plaquetas , Plaquetas , Retardo do Crescimento FetalRESUMO
Bone remodeling mediated by bone-forming osteoblasts (OBs) and bone-resorbing osteoclasts (OCs) maintains bone structure and function. Excessive OC activation leads to bone-destroying diseases such as osteoporosis and bone erosion of rheumatoid arthritis (RA). Differentiation of OCs from bone marrow cells (BMCs) is regulated by the bone microenvironment. The proinflammatory cytokine interleukin (IL)-1ß reportedly enhances osteoclastogenesis and plays important roles in RA-associated bone loss. The present study investigated the effect of IL-1ß on OC formation via microenvironmental cells. Treating mouse BMCs with IL-1ß in the presence of receptor activator of NF-κB ligand and macrophage colony-stimulating factor increased the number of OCs. Real-time RT-PCR revealed increased expression of the IL-1ß, IL-1RI, and IL-1RII genes in non-OCs compared with OCs. Removing CD45- cells which cannot differentiate into OCs, from mouse BMCs reduced the IL-1ß-mediated enhancement of osteoclastogenesis. IL-1ß treatment upregulated the expression of inducible nitric oxide synthase, insulin-like growth factor 2 (IGF2), and the chemokines stromal cell derived factor 1, C-X3-C motif ligand 1 (CX3CL1), and CXCL7 in non-OCs. Neutralizing antibodies against these chemokines and IGF2 suppressed osteoclastogenesis in the presence of IL-1ß. These results suggest that IL-1ß enhances osteoclastogenesis by upregulating IGF2 and chemokine expression in non-OCs.
Assuntos
Osteoclastos , Osteogênese , Camundongos , Animais , Osteogênese/genética , Ligantes , Células Cultivadas , Osteoclastos/metabolismo , Osteoblastos/metabolismo , Diferenciação Celular/genética , Ligante RANK/genética , Ligante RANK/metabolismoRESUMO
The tumor suppressor p53 is a transcription factor that regulates the expression of dozens of target genes and diverse physiological processes. To precisely regulate the p53 network, p53 undergoes various post-translational modifications and alters the selectivity of target genes. Acetylation plays an essential role in cell fate determination through the activation of p53. Although the acetylation of p53 has been examined, the underlying regulatory mechanisms remain unclear and, thus, have attracted the interest of researchers. We herein discuss the role of acetylation in the p53 pathway, with a focus on p53 acetyltransferases and deacetylases. We also review recent findings on the regulators of these enzymes to understand the mode of p53 acetylation from a broader perspective.
Assuntos
Processamento de Proteína Pós-Traducional , Proteína Supressora de Tumor p53 , Proteína Supressora de Tumor p53/metabolismo , Acetilação , Acetiltransferases/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Osteoporosis is caused by an imbalance in bone remodeling due to abnormal osteoclast (OC) formation and activation. Hypoxia at the site of inflammation promotes OC formation and activation in various species, including humans. We previously reported that insulin-like growth factor 2 (IGF2) plays an important role in osteoclastogenesis under hypoxia. In our present study, we focused on the mechanism of osteoclastogenesis in regard to IGF2 signaling under hypoxia. We confirmed that the addition of IGF2 promoted osteoclastogenesis under normoxic conditions. Conversely, IGF2-neutralizing antibodies inhibited osteoclastogenesis under both normoxic and hypoxic conditions. IGF2 addition increased levels of phosphorylated Akt (Thr308 and Ser473) and NF-κB (Ser536), indicating activation of the Akt-NF-κB pathway. IGF2 also increased the expression of inducible nitric oxide synthase, which promotes osteoclastogenesis via nitric oxide production. Expression levels of genes encoding inflammatory cytokines, such as tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6, were upregulated, indicating that IGF2 promotes osteoclastogenesis by increasing the expression of inflammatory cytokines via activation of the Akt-NF-κB pathway. These results suggest that IGF2 is a promising therapeutic target for osteoporosis and rheumatoid arthritis.
Assuntos
Citocinas , Hipóxia , Fator de Crescimento Insulin-Like II , Osteogênese , Citocinas/metabolismo , Humanos , Fator de Crescimento Insulin-Like II/metabolismo , NF-kappa B/metabolismo , Osteoporose , Proteínas Proto-Oncogênicas c-aktRESUMO
Photodynamic therapy (PDT) damages cancer cells via photosensitization using harmless laser irradiation. We synthesized a new photosensitizer, mannose-conjugated-chlorin e6 (M-chlorin e6), which targets mannose receptors that are highly expressed on M2-like tumor-associated macrophages (M2-TAMs) and cancer cells. In our previous study, we demonstrated that M-chlorin e6 PDT reduces tumor volume and decreases the proportion of M2-TAMs. Whether M-chlorin e6 PDT-treated cancer cells activate tumor immunity remains unclear, although the decrease in M2-TAMs is thought to be a direct injurious effect of M-chlorin e6 PDT. Calreticulin (CRT) is exposed at the surface of the membrane of cancer cells in response to treatment with chemotherapeutic agents such as anthracycline and oxaliplatin. Surface-exposed CRT induces phagocytosis of CRT receptor-positive cells, including macrophages, inducing anticancer immune responses. In the present study, we found that M-chlorin e6 PDT increases CRT on the surface of cancer cells, leading to macrophage phagocytosis of cancer cells. Furthermore, M-chlorin e6 PDT increases CD80+CD86+ macrophages. These results suggest that M-chlorin e6 PDT exerts anti-tumor effects by both enhancing the phagocytosis of cancer cells and strengthening the anti-tumor phenotype of macrophages.
Assuntos
Clorofilídeos , Neoplasias , Fotoquimioterapia , Calreticulina , Clorofilídeos/uso terapêutico , Humanos , Macrófagos , Manose/farmacologia , Manose/uso terapêutico , Neoplasias/tratamento farmacológico , Fagocitose , Fotoquimioterapia/métodosRESUMO
The proximal tubules, which are part of the kidney, maintain blood homeostasis by absorbing amino acids, glucose, water, and ions such as sodium (Na), potassium, and bicarbonate. Proximal tubule dysfunction is associated with the pathogenesis of many kidney diseases. Renal proximal tubular epithelial cells (RPTECs) are responsible for the main functions of the proximal tubules. Therefore, in vitro experiments using RPTECs would greatly enhance our understanding of nephron physiology and pathobiology. It is preferable to use immortalized cell lines, such as human kidney-2 (HK-2) cells, because they are derived from humans and maintain growth indefinitely. However, tissue-specific RPTEC phenotypes, including apical-basal polarization, are frequently lost in conventional two-dimensional culture methods in part due to microenvironmental deficiencies. To overcome this limitation, we developed a three-dimensional (3D) spheroid culture method for HK-2 cells using an extracellular matrix. HK-2 spheroids in 3D culture formed a tubule-like architecture with cellular polarity and showed markedly restored Na transport function. 3D culture of HK-2 cells also increased expression of kidney development-related genes, including WNT9B. Models of human renal tubules using HK-2 spheroids will greatly improve our understanding of the physiology and pathobiology of the kidney.
Assuntos
Polaridade Celular/fisiologia , Células Epiteliais/citologia , Túbulos Renais Proximais/citologia , Túbulos Renais/metabolismo , Transporte Biológico , Linhagem Celular , Matriz Extracelular/metabolismo , Humanos , Rim/metabolismo , Sódio/metabolismoAssuntos
Fígado/fisiopatologia , Trombocitopenia/fisiopatologia , Trombopoetina/metabolismo , Animais , Animais Recém-Nascidos , Peso ao Nascer , Feminino , Hipertensão/complicações , Fígado/metabolismo , Gravidez , Complicações Cardiovasculares na Gravidez/etiologia , Ratos , Ratos Wistar , Trombocitopenia/etiologia , Trombocitopenia/metabolismo , Trombopoetina/análiseRESUMO
M2-like tumor-associated macrophages (M2-TAMs) in cancer tissues are intimately involved in cancer immunosuppression in addition to growth, invasion, angiogenesis, and metastasis. Hence, considerable attention has been focused on cancer immunotherapies targeting M2-TAMs. However, systemic therapies inhibit TAMs as well as other macrophages important for normal immune responses throughout the body. To stimulate tumor immunity with fewer side effects, we targeted M2-TAMs using photodynamic therapy (PDT), which damages cells via a nontoxic photosensitizer with harmless laser irradiation. We synthesized a light-sensitive compound, mannose-conjugated chlorin e6 (M-chlorin e6), which targets mannose receptors highly expressed on M2-TAMs. M-chlorin e6 accumulated more in tumor tissue than normal skin tissue of syngeneic model mice and was more rapidly excreted than the second-generation photosensitizer talaporfin sodium. Furthermore, M-chlorin e6 PDT significantly reduced the volume and weight of tumor tissue. Flow cytometric analysis revealed that M-chlorin e6 PDT decreased the proportion of M2-TAMs and increased that of anti-tumor macrophages, M1-like TAMs. M-chlorin e6 PDT also directly damaged and killed cancer cells in vitro. Our data indicate that M-chlorin e6 is a promising new therapeutic agent for cancer PDT.
RESUMO
Atherosclerosis is an important underlying cause of cardiovascular diseases; vascular endothelial cells play a vital role in inflammatory responses in the initial steps of atherosclerosis. High levels of the pro-inflammatory cytokine interleukin-6 (IL-6) long have been considered a risk factor in the development and complications of atherosclerotic disease. However, it is still controversial whether IL-6 is atherogenic or atheroprotective. Recently, miR-126-3p, an endothelial cell-specific microRNA, has been proposed as an atheroprotective molecule. Therefore, we investigated whether IL-6 accelerates endothelial cell responses through the suppression of miR-126-3p expression in human endothelial cell line EA.hy926. IL-6 yielded concentration-dependent decreases in miRNA-126-3p accumulation in EA.hy926 cells, leading in turn to increased expression of genes targeted by miRNA-126-3p. In addition, adhesion of the human monocyte cell line THP-1 was enhanced by the exposure of EA.hy926 cells to IL-6, with associated increases in the levels of the adhesion molecule intercellular adhesion molecule-1 (ICAM-1). Suppression of miR-126-3p expression resulted in upregulation of miRNA-126-3p-regulated genes, enhanced adhesion of THP-1 cells, and increased ICAM-1 accumulation in EA.hy926 cells. In contrast, miR-126-3p overproduction had the opposite effects. The regulation of miRNA-126-3p by IL-6 may have important implications for the development of novel protective therapies targeting atherosclerosis.
Assuntos
Interleucina-6/metabolismo , Interleucina-6/farmacologia , MicroRNAs/efeitos dos fármacos , MicroRNAs/genética , Aterosclerose/tratamento farmacológico , Aterosclerose/metabolismo , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/efeitos dos fármacos , Molécula 1 de Adesão Intercelular/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Regulação para CimaRESUMO
BACKGROUND: In vitro blood-brain barrier (BBB) models using human induced pluripotent stem (iPS) cell-derived brain microvascular endothelial-like cells (iBMELCs) have been developed to predict the BBB permeability of drug candidates. For the differentiation of iBMELCs, Matrigel, which is a gelatinous protein mixture, is often used as a coating substrate. However, the components of Matrigel can vary among lots, as it is obtained from mouse sarcoma cells with the use of special technics and also contains various basement membranes. Therefore, fully defined substrates as substitutes for Matrigel are needed for a stable supply of iBMELCs with less variation among lots. METHODS: iBMELCs were differentiated from human iPS cells on several matrices. The barrier integrity of iBMELCs was evaluated based on transendothelial electrical resistance (TEER) values and permeability of fluorescein isothiocyanate-dextran 4 kDa (FD4) and Lucifer yellow (LY). Characterization of iBMELCs was conducted by RT-qPCR and immunofluorescence analysis. Functions of efflux transporters were defined by intracellular accumulation of the substrates in the wells of multiwell plates. RESULTS: iBMELCs differentiated on laminin 221 fragment (LN221F-iBMELCs) had higher TEER values and lower permeability of LY and FD4 as compared with iBMELCs differentiated on Matrigel (Matrigel-iBMELCs). Besides, the gene and protein expression levels of brain microvascular endothelial cells (BMEC)-related markers were similar between LN221F-iBMELCs and Matrigel-iBMELCs. Moreover, both Matrigel- and LN221F-iBMELCs had functions of P-glycoprotein and breast cancer resistance protein, which are essential efflux transporters for barrier functions of the BBB. CONCLUSION: The fully defined substrate LN221F presents as an optimal coating matrix for differentiation of iBMELCs. The LN221F-iBMELCs had more robust barrier function for a longer period than Matrigel-iBMELCs with characteristics of BMECs. This finding will contribute the establishment of an iBMELC supply system for pharmacokinetic and pathological models of the BBB.
Assuntos
Materiais Biocompatíveis , Barreira Hematoencefálica/fisiologia , Diferenciação Celular/fisiologia , Colágeno , Células Endoteliais/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Laminina , Microvasos/citologia , Proteoglicanas , Animais , Barreira Hematoencefálica/citologia , Linhagem Celular , Células Cultivadas , Técnicas Citológicas , Combinação de Medicamentos , Células Endoteliais/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , CamundongosRESUMO
BACKGROUND: We previously discovered that renal macrophages (Mφs) phagocytose renal calcium oxalate monohydrate (COM) crystals. This study investigated the processing of engulfed crystals using in vitro models. METHODS: J774.1 mouse Mφs were exposed to COM crystals and observed for 24 h using polarized light microscopy with/without cytochalasin B (CB), an inhibitor of phagocytosis, to confirm active crystal phagocytosis. LysoTracker and immunohistochemical staining using transmission electron microscopy for lysosomal-associated membrane protein 1 were used to confirm engulfed COM crystal uptake into lysosomes. Diachronic tracking of specific Mφs was performed to capture the entire course of engulfed COM crystal processing using polarized light microscopy. Follow-up studies of fluorescent COM (f-COM) crystals using imaging cytometry were performed in the presence and absence of nigericin to dissipate the pH gradient in acidic organelles. RESULTS: Phagocytosis rates increased with COM density and were significantly lower in cells treated with CB (p < 0.01). We observed that engulfed crystals colocalized within lysosomes of the Mφs; moreover, diachronic observation indicated that the engulfed COM crystals were subdivided during Mφ division and eliminated by the 7th day of culture. Additionally, imaging cytometry showed that the fluorescence level of f-COM crystals in the nigericin (-) group after 48 h was significantly lower than that in the nigericin (+) group. CONCLUSIONS: This study confirmed active phagocytosis and lysosomal processing of engulfed COM crystals by Mφs. This discovery is expected to contribute to the development of future drugs that enhance the COM crystal phagocytic ability of Mφs.
Assuntos
Oxalato de Cálcio/metabolismo , Macrófagos/metabolismo , Fagocitose/fisiologia , Animais , Cristalização , Modelos Animais de Doenças , Humanos , Técnicas In Vitro , CamundongosRESUMO
Transplanted endothelial progenitor cells (EPCs) repair blood vessels and exert regenerative effects on disorders such as lower limb ischemia. EPCs serve as a model for pathophysiological and pharmacokinetic studies, which is important for drug discovery. However, primary human EPCs are phenotypically unstable, which limits their clinical utility. Therefore, we employed human induced pluripotent stem (iPS) cells to circumvent this problem. Here we focused on human iPS cell-derived sac-like structures (iPS-sacs), which contain endothelial lineage cells and hematopoietic lineage cells. Previous studies isolated only hematopoietic lineage cells from iPS-sacs. Therefore, here we attempted to isolate EPCs. However, iPS-sacs generated by a published protocol did not contain sufficient EPCs. Therefore, to generate iPS-sacs highly enriched in EPCs, we added the glycogen synthase kinase 3 beta (GSK3ß) inhibitor CHIR-99021 to the culture medium early during differentiation. The cells rapidly differentiated into mesoderm to yield abundant EPCs, and CHIR-99021 increased the proportion of EPCs contained in iPS-sacs. EPCs, which were purified using anti-platelet endothelial cell adhesion molecule (PECAM1) antibody-conjugated beads, expressed markers of immature endothelial cells. Purified EPCs formed tube-like structures and incorporated acetylated low density lipoprotein (Ac-LDL), reflecting endothelial phenotypes. The simple method described here will likely improve regenerative medicine and facilitate basic studies on the endothelial lineage.