[Show abstract][Hide abstract] ABSTRACT: Chronic obstructive pulmonary disease (COPD) shows progressive, irreversible airflow limitation induced by emphysema and lung inflammation. The aim of the present study was to determine if COPD conditions induce blood-brain barrier (BBB) dysfunction. We found that the intratracheal administration of porcine pancreatic elastase (PPE; 3 U) induced alveolar enlargement, increased neutrophil number in bronchoalveolar lavage fluid, and decreased blood oxygen saturation in mice at 21 days after inhalation. In parallel with these lung damages, BBB permeability to sodium fluorescein and Evans blue albumin was markedly increased. Our findings demonstrate that COPD conditions are associated with risk for BBB impairment.
[Show abstract][Hide abstract] ABSTRACT: Tight junctions (TJs) of the epidermis play an important role in maintaining the epidermal barrier. TJ breakdown is associated with skin problems, such as wrinkles and transepidermal water loss (TEWL). Clinical studies have reported that topical nifedipine is effective in reducing the depth of wrinkles and improving TEWL. However, it remains unknown whether nifedipine influences the TJ function in the epidermis. In the present study, we investigated the effect of nifedipine on epidermal barrier dysfunction in normal human epidermal keratinocytes (NHEKs) treated with sodium caprate (C10), a TJ inhibitor. Nifedipine reversed the C10-decreased transepithelial electrical resistance values as a measure of disruption of the epidermal barrier. Immunocytochemical observations revealed that nifedipine improved the C10-induced irregular arrangement of claudin-1, a key protein in TJs. Taken together, these findings suggest that nifedipine prevents epidermal barrier dysfunction, at least in part, by reconstituting the irregular claudin-1 localization at TJs in C10-treated NHEKs.
[Show abstract][Hide abstract] ABSTRACT: Varenicline is one of the most widely used drugs for smoking cessation. However, whether an adverse effect of varenicline is associated with the risk of serious cardiovascular events remains controversial. In this study, we determined if varenicline increases the risk of cardiovascular events using apolipoprotein E knockout (ApoE KO) mice. ApoE KO mice (8 weeks old) were injected with varenicline 0.5 mg kg(-1) day(-1) for 3 weeks. Varenicline aggravated atherosclerotic plaque formation in whole aorta from ApoE KO mice compared with vehicle. Methyllycaconitine, an alpha 7 nicotinic acetylcholine receptor (nAChR) antagonist, inhibited varenicline-induced aggravated plaque formation. Our findings show that varenicline progresses atherosclerotic plaque formation through alpha 7 nAChR, and thereby increases the risk of cardiovascular events. (C) 2014 The Authors. Published by Elsevier Inc.
Full-text · Article · Nov 2014 · Biochemical and Biophysical Research Communications
[Show abstract][Hide abstract] ABSTRACT: Background
Elevated level of reactive carbonyl species, such as methylglyoxal, triggers carbonyl stress and activates a series of inflammatory responses leading to accelerated vascular damage. Edaravone is the active substance of a Japanese medicine, which aids neurological recovery following acute brain ischemia and subsequent cerebral infarction. Our aim was to test whether edaravone can exert a protective effect on the barrier properties of human brain endothelial cells (hCMEC/D3 cell line) treated with methylglyoxal.
Cell viability was monitored in real-time by impedance-based cell electronic sensing. The barrier function of the monolayer was characterized by measurement of resistance and flux of permeability markers, and visualized by immunohistochemistry for claudin-5 and β-catenin. Cell morphology was also examined by holographic phase imaging.
Methylglyoxal exerted a time- and dose-dependent toxicity on cultured human brain endothelial cells: a concentration of 600 µM resulted in about 50% toxicity, significantly reduced the integrity and increased the permeability of the barrier. The cell morphology also changed dramatically: the area of cells decreased, their optical height significantly increased. Edaravone (3 mM) provided a complete protection against the toxic effect of methylglyoxal. Co-administration of edaravone restored cell viability, barrier integrity and functions of brain endothelial cells. Similar protection was obtained by the well-known antiglycating molecule, aminoguanidine, our reference compound.
These results indicate for the first time that edaravone is protective in carbonyl stress induced barrier damage. Our data may contribute to the development of compounds to treat brain endothelial dysfunction in carbonyl stress related diseases.
[Show abstract][Hide abstract] ABSTRACT: Brain pericytes are involved in neurovascular dysfunction, neurodegeneration and/or neuroinflammation. In the present study, we focused on the proinflammatory properties of brain pericytes to understand their participation in the induction of inflammation at the neurovascular unit (NVU). The NVU comprises different cell types, namely, brain microvascular endothelial cells, pericytes, astrocytes and microglia. Among these, we found pericytes to be the most sensitive to tumor necrosis factor (TNF)-α, possessing a unique cytokine and chemokine release profile. This was characterized by marked release of interleukin (IL)-6 and macrophage inflammatory protein-1α. Furthermore, TNF-α-stimulated pericytes induced expression of inducible nitric oxide synthase and IL-1β mRNAs, as an index of BV-2 microglial cell activation state, to the highest levels. Based on these findings, the possibility that brain pericytes act specifically as TNF-α-sensitive cells and as effectors of TNF-α through the release of proinflammatory factors, and that, as such, they have a role in inducing brain inflammation, should be considered.
No preview · Article · Jun 2014 · Neuroscience Letters
[Show abstract][Hide abstract] ABSTRACT: The main therapeutic strategy against human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) characterized by lower extremity motor dysfunction is immunomodulatory treatment, with drugs such as corticosteroid hormone and interferon-α, at present. However, there are many issues in long-term treatment with these drugs, such as insufficient effects and various side effects. We now urgently need to develop other therapeutic strategies. The heparinoid, pentosan polysulfate sodium (PPS), has been safely used in Europe for the past 50 years as a thrombosis prophylaxis and for the treatment of phlebitis. We conducted a clinical trial to test the effect of subcutaneous administration of PPS in 12 patients with HAM/TSP in an open-labeled design. There was a marked improvement in lower extremity motor function, based on reduced spasticity, such as a reduced time required for walking 10 m and descending a flight of stairs. There were no significant changes in HTLV-I proviral copy numbers in peripheral blood contrary to the inhibitory effect of PPS in vitro for intercellular spread of HTLV-I. However, serum soluble vascular cell adhesion molecule (sVCAM)-1 was significantly increased without significant changes of serum level of chemokines (CXCL10 and CCL2). There was a positive correlation between increased sVCAM-1and reduced time required for walking 10 m. PPS might induce neurological improvement by inhibition of chronic inflammation in the spinal cord, through blocking the adhesion cascade by increasing serum sVCAM-1, in addition to rheological improvement of the microcirculation. PPS has the potential to be a new therapeutic tool for HAM/TSP.
No preview · Article · Mar 2014 · Journal of NeuroVirology
[Show abstract][Hide abstract] ABSTRACT: To investigate the molecular mechanisms of lung cancer-induced bone metastasis, we established a bone-seeking subclone (HARA-B4) from a human squamous lung cancer cell line (HARA) using an in vivo selection method. We compared comprehensive gene expression profiles between HARA and HARA-B4, and identified the critical factors for the formation of bone metastasis using in vitro and in vivo assays. The number of bone metastatic colonies in the hind legs was significantly higher in HARA-B4-inoculated mice than in HARA-inoculated mice at 4 weeks after inoculation. In addition, visceral (adrenal) metastases were not found in HARA-B4-inoculated mice at autopsy, suggesting an increase in cancer cell tropism to bone in HARA-B4. Based on a comprehensive gene expression analysis, the expression level of CXC chemokine ligand 14 (CXCL14) was 5-fold greater in HARA-B4 than in HARA. Results of a soft agar colony formation assay showed that anchorage-independent growth ability was 4.5-fold higher with HARA-B4 than with HARA. The murine pre-osteoblast cell line MC3T3-E1 and the pre-osteoclast/macrophage cell line RAW264.7 migrated faster toward cultured HARA-B4 cells than toward HARA cells in a transwell cell migration assay. Interestingly, CXCL14 was shown to be involved in all events (enhancement of cancer cell tropism to the bone, anchorage-independent growth and/or recruitment of bone marrow cells) based on siRNA experiments in HARA-B4 cells. Furthermore, in clinical specimens of lung cancer-induced bone metastasis, expression of CXCL14 was observed in the tumor cells infiltrated in bone marrow in all specimens examined. CXCL14 was able to promote bone metastasis through enhancement of cancer cell tropism to the bone and/or recruitment of bone marrow cells around metastatic cancer cells.
Preview · Article · Feb 2014 · International Journal of Oncology
[Show abstract][Hide abstract] ABSTRACT: Combined treatment with cyclosporine microemulsion preconcentrate (CyA MEPC) and steroids has been widely used for idiopathic membranous nephropathy (IMN) associated with steroid-resistant nephrotic syndrome (SRNS). Recent studies have shown that once-a-day and preprandial administration of CyA MEPC is more advantageous than the conventional twice-a-day administration in achieving the target blood CyA concentration at 2 h post dose (C2). We designed a randomized trial to compare these administrations.
IMN patients with SRNS (age 16-75 years) were divided prospectively and randomly into 2 groups. In group 1 (n = 23), 2-3 mg/kg body weight (BW) CyA MEPC was given orally once a day before breakfast. In group 2 (n = 25), 1.5 mg/kg BW CyA MEPC was given twice a day before meals. CyA + prednisolone was continued for 48 weeks.
Group 1 showed a significantly higher cumulative complete remission (CR) rate (p = 0.0282), but not when incomplete remission 1 (ICR1; urine protein 0.3-1.0 g/day) was added (p = 0.314). Because a C2 of 600 ng/mL was determined as the best cut-off point, groups 1 and 2 were further divided into subgroups A (C2 ≥600 ng/mL) and B (C2 <600 ng/mL). Groups 1A and 2A revealed significantly higher cumulative remission (CR + ICR1) (p = 0.0069) and CR-alone (p = 0.0028) rates. On the other hand, 3 patients with high CyA levels (C2 >900 ng/mL) in Group 1A were withdrawn from the study because of complications.
CyA + prednisolone treatment is effective for IMN with associated SRNS at a C2 of ≥600 ng/mL. To achieve remission, preprandial once-a-day administration of CyA at 2-3 mg/kg BW may be the most appropriate option. However, we should adjust the dosage of CyA by therapeutic drug monitoring to avoid complications.
Full-text · Article · Dec 2013 · Clinical and Experimental Nephrology
[Show abstract][Hide abstract] ABSTRACT: BMP4, a member of the transforming growth factor-beta superfamily, is upregulated in the aortas of diabetic db/db mice. However, little is known about its role in diabetic atherosclerosis. Therefore, we examined the roles of BMP4 in the formation of diabetic atherosclerosis in apolipoprotein E knockout (ApoE KO) mice and in the uptake of oxidized low density lipoprotein (oxLDL) in peritoneal macrophages of wild-type mice.
To induce diabetes, ApoE KO mice were intraperitoneally injected with streptozotocin. Diabetic and non-diabetic ApoE KO mice were then fed a high-fat diet for 4 weeks. Next, to investigate a role of BMP4 in the peritoneal macrophages, we examined the uptake of oxLDL in BMP4-treated macrophages.
Diabetic ApoE KO mice showed accelerated progression of aortic plaques accompanied by increased luminal plaque area. Western blot analysis showed that BMP4 expression in the whole aorta was greatly increased in diabetic ApoE KO mice, than non-diabetic mice. Western blot analysis showed that the BMP4/SMAD1/5/8 signaling pathway was strongly activated in the aorta from diabetic ApoE KO mice, compared with control ApoE KO mice. Double immunofluorescence staining showed that BMP4 is expressed in MOMA2-labeled macrophage in the aortic lesions of ApoE KO mice. BMP4 significantly increased the uptake of oxLDL into peritoneal macrophages in vitro.
We show that in the aorta of diabetic ApoE KO mice, BMP4 is increased and activates SMAD1/5/8. Our in vitro findings indicate that BMP4 enhances oxLDL uptake in mouse peritoneal macrophages, suggesting BMP4 may be involved in aortic plaque formation in diabetic ApoE KO mice. Targeting BMP4 may offer a new strategy for inhibition of plaque progression and stabilization of atherosclerotic lesions.
Full-text · Article · Oct 2013 · Journal of Inflammation
[Show abstract][Hide abstract] ABSTRACT: Blood-brain barrier (BBB) disruption occurs frequently in CNS diseases and injuries. Few drugs have been developed as therapeutic candidates for facilitating BBB functions. Here, we examined whether metformin up-regulates BBB functions using rat brain microvascular endothelial cells (RBECs). Metformin, concentration- and time-dependently increased transendothelial electrical resistance of RBEC monolayers, and decreased RBEC permeability to sodium fluorescein and Evans blue albumin. These effects of metformin were blocked by compound C, an inhibitor of AMP-activated protein kinase (AMPK). AMPK stimulation with an AMPK activator, AICAR, enhanced BBB functions. These findings indicate that metformin induces up-regulation of BBB functions via AMPK activation.
No preview · Article · Mar 2013 · Biochemical and Biophysical Research Communications
[Show abstract][Hide abstract] ABSTRACT: The blood-brain barrier (BBB) is formed by brain endothelial cells. Many immortalized brain endothelial cell lines have been established; these have been used as in vitro BBB models. The aim of the present study was to assess the paracellular barrier properties of the immortalized mouse brain endothelial cell lines bEND.3, bEND.5 cells, and mouse brain endothelial cell 4 (MBEC4), and those of the primary mouse brain endothelial cells pMBECs. bEND.3 cells showed low permeability to sodium fluorescein and obvious staining of tight junction proteins (claudin-5, occludin and ZO-1) similar to pMBECs; these barrier properties of MBEC4 and bEND.5 cells were low. In addition, bEND.3 cells expressed the highest level of claudin-5 among all cells. These results suggest that bEND.3 cells are a convenient and useful model for evaluating BBB function, especially the paracellular barrier.
No preview · Article · Mar 2013 · Biological & Pharmaceutical Bulletin
[Show abstract][Hide abstract] ABSTRACT: The blood-brain barrier (BBB) restricts the entry of circulating drugs and xenobiotics into the brain, and thus its permeability to substances is a critical factor that determines their central effects. The infant brain is vulnerable to neurotoxic substances partly due to the immature BBB. The employment of in vitro BBB models to evaluate permeability of compounds provides higher throughput than that of in vivo animal experiments. However, existing in vitro BBB models have not been able to simulate the intrinsic neonatal BBB. To establish a neonatal BBB model that mimics age-related BBB properties, the neonatal and adult in vitro BBB models were constructed with brain endothelial cells isolated from 2- and 8-week-old rats, respectively. To evaluate BBB functions, transendothelial electrical resistance, permeability of sodium fluorescein and Evans blue-albumin, and transport of rhodamine123 were measured. Radiolabelled drugs were used for BBB permeability studies in the neonatal and adult BBB models (in vitro) and in age-matched rats (in vivo). The neonatal BBB model showed lower barrier and p-glycoprotein (P-gp) functions than the adult BBB model; these were well associated with lower expressions of the barrier-related proteins and P-gp, and a different distribution pattern of immunostained barrier-related proteins. Verapamil (a P-gp inhibitor) significantly increased the influx of rhodamine 123, supporting functional P-gp expression in the neonatal BBB model. Valproic acid, but not nicotine, showed higher BBB permeability in the neonatal BBB model, which was well in accordance with the in vivo BBB property. We established a neonatal BBB model in vitro. This could allow us to assess the age-dependent BBB permeability of drugs.
[Show abstract][Hide abstract] ABSTRACT: Background
Cyclophilin A (CypA), a member of the immunophilin family, is a ubiquitously distributed intracellular protein. Recent studies have shown that CypA is secreted by cells in response to inflammatory stimuli. Elevated levels of extracellular CypA and its receptor, CD147 have been detected in the synovium of patients with RA. However, the precise process of interaction between CypA and CD147 in the development of RA remains unclear. This study aimed to investigate CypA secretion from fibroblast-like synoviocytes (FLS) isolated from mice with collagen-induced arthritis (CIA) and CypA-induced CD147 expression in mouse macrophages.
CIA was induced by immunization with type II collagen in mice. The expression and localization of CypA and CD147 was investigated by immunoblotting and immunostaining. Both CypA and CD147 were highly expressed in the joints of CIA mice. CD147 was expressed in the infiltrated macrophages in the synovium of CIA mice. In vitro, spontaneous CypA secretion from FLS was detected and this secretion was increased by stimulation with lipopolysaccharide. CypA markedly increased CD147 levels in macrophages.
These findings suggest that an interaction in the synovial joints between extracellular CypA and CD147 expressed by macrophages may be involved in the mechanisms underlying the development of arthritis.
Full-text · Article · Nov 2012 · Journal of Inflammation
[Show abstract][Hide abstract] ABSTRACT: Recently, 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) reductase inhibitors were reported to induce neurite outgrowth in vitro. However, the mechanism underlying this effect remains unclear. Cellular prion protein (PrP(C)) is a ubiquitous glycoprotein present on the surfaces of various cells, including neurons, and is suggested to be involved in neurite outgrowth. Therefore, the present study aimed to determine whether PrP(C) mediates neurite outgrowth induced by HMG-CoA reductase inhibitors. Atorvastatin, a strong HMG-CoA reductase inhibitor, induced neurite outgrowth and increased PrP(C) levels in Neuro2a cells in a time- and dose-dependent manner. PrP(C) mRNA expression was also increased by atorvastatin. Farnesol, a non-sterol mevalonate derivative, attenuated the atorvastatin-induced neurite outgrowth and increase in PrP(C). Neuro2a cells overexpressing PrP(C) showed a remarkable enhancement of atorvastatin-induced neurite outgrowth compared with mock cells transfected with empty pCI-neo vector. These findings suggest that PrP(C) contributes, at least in part, to atorvastatin-induced neurite outgrowth. This phenomenon may be included among the mechanisms underlying decreased risk of Alzheimer's disease in patients treated with HMG-CoA reductase inhibitors.
No preview · Article · Nov 2012 · Neuroscience Letters