Search

Background Given that cathepsin S (CatS) gained attention due to its enzymatic and non-enzymatic functions in signaling, the role of CatS in ischemia-induced angiogenesis of aged mice was explored.Methods and Results:To study the role of CatS in the decline in aging-related vascular regeneration capacity, a hindlimb ischemia model was applied to aged wild-type (CatS+/+) and CatS-deficient (CatS-/-) mice. CatS-/-mice exhibited impaired blood flow recovery and capillary formation and increased levels of p-insulin receptor substrate-1, Wnt5a, and SC35 proteins and decreased levels of phospho-endothelial nitric oxide synthase (p-eNOS), p-mTOR, p-Akt, p-ERK1/2, p-glycogen synthase kinase-3α/β, and galatin-3 proteins, as well as decreased macrophage infiltration and matrix metalloproteinase-2/-9 activities in the ischemic muscles. In vitro, CatS knockdown altered the levels of these targeted essential molecules for angiogenesis. Together, the results suggested that CatS-/-leads to defective endothelial cell functions and that CatS-/-is associated with decreased circulating endothelial progenitor cell (EPC)-like CD31+/c-Kit+cells. This notion was reinforced by the study finding that pharmacological CatS inhibition led to a declined angiogenic capacity accompanied by increased Wnt5a and SC35 levels and decreased eNOS/Akt-ERK1/2 signaling in response to ischemia. Conclusions These findings demonstrated that the impairment of ischemia-induced neovascularization in aged CatS-/-mice is due, at least in part, to the attenuation of endothelial cell/EPC functions and/or mobilization associated with Wnt5a/SC35 activation in advanced age.

Objective Exposure to chronic psychosocial stress is a risk factor for metabolic cardiovascular disorders. Given that dipeptidyl peptidase-4 (DPP-4) has an important role in human pathobiology, we investigated the role of DPP-4 in stress-related thrombosis in mice, focusing on oxidative stress and the von Willebrand factor (vWF)-cleaving protease ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13). Methods and results Male mice randomly assigned to nonstress and 2-week immobilized-stress groups underwent iron chloride3 (FeCl3)-induced carotid artery thrombosis surgery for morphological and biochemical studies at specific times. On day 14 post-stress/surgery, stress had enhanced the lengths and weights of arterial thrombi, with alterations of plasma DPP-4, plasminogen activation inhibitor-1 and ADAMTS13. The stressed mice had increased levels of vascular cell adhesion molecule-1, intracellular adhesion molecule-1, monocyte chemoattractant protein-1, gp91phox, p22phox, matrix metalloproteinase-2 (MMP-2), MMP-9, cathepsins S and K mRNAs and/or proteins, and reduced levels of endothelial nitric oxide synthase, catalase and superoxide dismutase-1 mRNAs and/or proteins. Stress also accelerated arterial endothelial cell damage. The DPP-4 inhibitor anagliptin ameliorated the stress-induced targeted molecular and morphological changes and thrombosis. In vitro, DPP-4 inhibition also mitigated the alterations in the targeted ADAMTS13 and other oxidative and inflammatory molecules in human umbilical vein endothelial cells in response to H2O2. Conclusion DPP-4 inhibition appeared to improve the FeCl3-induced thrombosis in mice that received stress, possibly via the improvement of ADAMTS13 and oxidative stress, suggesting that DPP-4 could become a novel therapeutic target for chronic psychological stress-related thrombotic events in metabolic cardiovascular disorders.

Background Chronic psychological stress (CPS) is linked to cardiovascular disease initiation and progression. Given that cysteinyl cathepsin K (CatK) participates in vascular remodeling and atherosclerotic plaque growth in several animal models, we investigated the role of CatK in the development of experimental neointimal hyperplasia in response to chronic stress. Methods and results At first, male wild-type (CatK) mice that underwent carotid ligation injury were subjected to chronic immobilization stress. On postoperative and stressed day 14, the results demonstrated that stress accelerated injury-induced neointima hyperplasia. On day 4, stressed mice showed following: increased levels of monocyte chemoattractant protein-1, gp91phox, toll-like receptor-2 (TLR2), TLR4, and CatK mRNAs or/and proteins, oxidative stress production, aorta-derived smooth muscle cell (SMC) migration, and macrophage infiltration as well as targeted intracellular proliferating-related molecules. Stressed mice showed increased matrix metalloproteinase-2 (MMP-2) and MMP-9 mRNA expressions and activities and elastin disruption in the injured carotid arteries. Second, CatK and CatK deficiency (CatK) mice received ligation injury and stress to explore the role of CatK. The stress-induced harmful changes were prevented by CatK. Finally, CatK mice that had undergone ligation surgery were randomly assigned to one of two groups and administered vehicle or CatK inhibitor for 14 days. Pharmacological CatK intervention produced a vascular benefit. Conclusion These data indicate that CatK deletion protects against the development of experimental neointimal hyperplasia via the attenuation of inflammatory overaction, oxidative stress production, and VSMC proliferation, suggesting that CatK is a novel therapeutic target for the management of CPS-related restenosis after intravascular intervention therapies.

Background Cathepsin K (CatK) is a widely expressed cysteine protease that has gained attention because of its enzymatic and non-enzymatic functions in signalling. Here, we examined whether CatK-deficiency (CatK−/−) would mitigate injury-related skeletal muscle remodelling and fibrosis in mice, with a special focus on inflammation and muscle cell apoptosis. Methods Cardiotoxin (CTX, 20 μM/200 μL) was injected into the left gastrocnemius muscle of male wild-type (CatK+/+) and CatK−/− mice, and the mice were processed for morphological and biochemical studies. Results On post-injection Day 14, CatK deletion ameliorated muscle interstitial fibrosis and remodelling and performance. At an early time point (Day 3), CatK−/− reduced the lesion macrophage and leucocyte contents and cell apoptosis, the mRNA levels of monocyte chemoattractant protein-1, toll-like receptor-2 and toll-like receptor-4, and the gelatinolytic activity related to matrix metalloproteinase-2/-9. CatK deletion also restored the protein levels of caspase-3 and cleaved caspase-8 and the ratio of the BAX to the Bcl-2. Moreover, CatK deficiency protected muscle fibre laminin and desmin disorder in response to CTX injury. These beneficial muscle effects were mimicked by CatK-specific inhibitor treatment. In vitro experiments demonstrated that pharmacological CatK inhibition reduced the apoptosis of C2C12 mouse myoblasts and the levels of BAX and caspase-3 proteins induced by CTX. Conclusions These results demonstrate that CatK plays an essential role in skeletal muscle loss and fibrosis in response to CTX injury, possibly via a reduction of inflammation and cell apoptosis, suggesting a novel therapeutic strategy for the control of skeletal muscle diseases by regulating CatK activity.

Background and aims Exposure to psychosocial stress is a risk factor for cardiovascular disorders. Because the glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) agonist prevents cardiovascular injury, we investigated the beneficial effects and mechanism of the GLP-1 analogue exenatide on stress-related vascular senescence and atherosclerosis in apolipoprotein E-deficient ( ApoE −/− ) mice fed a high-fat (HF) diet. Methods ApoE −/− mice fed the HF diet were assigned to non-stressed and immobilized-stress groups for 12 weeks. Mice fed the HF diet were divided into 2 groups and administered vehicle or exenatide for 12 weeks under stress conditions. Results Chronic stress enhanced vascular endothelial senescence and atherosclerotic plaque growth. The stress increased the levels of plasma depeptidyl peptidase-4 activity and decreased the levels of plasma GLP-1 and both plasma and adipose adiponectin (APN). As compared with the mice subjected to stress alone, the exenatide-treated mice had decreased plaque microvessel density, macrophage accumulation, broken elastin, and enhanced plaque collagen volume, and lowered levels of peroxisome proliferator-activated receptor-α, gp91phox osteopontin, C-X-C chemokine receptor-4, toll-like receptor-2 (TLR2), TLR4, and cathepsins K, L, and S mRNAs and/or proteins. Exenatide reduced aortic matrix metalloproteinase-9 ( MMP-9 ) and MMP-2 gene expression and activities. Exenatide also stimulated APN expression of preadipocytes and inhibited ox-low density lipoprotein-induced foam cell formation of monocytes in stressed mice. Conclusions These results indicate that the exenatide-mediated beneficial vascular actions are likely attributable, at least in part, to the enhancement of APN production and the attenuation of plaque oxidative stress, inflammation, and proteolysis in ApoE −/− mice under chronic stress.

Objectives Exposure to psychosocial stress is a risk factor for cardiovascular disease. Given that dipeptidyl peptidase-4 (DPP4) regulates several intracellular signaling pathways associated with glucagon-like peptide-1 (GLP-1) metabolism, we investigated the role of DPP4 in stress-related vascular senescence and atherosclerosis in apolipoprotein E -deficient (ApoE −/− ) mice. Methods and results ApoE −/− mice fed a high-fat (HF) diet were randomly assigned to one of non-stress and immobilized stress groups for 12weeks. Chronic stress accelerated vascular senescence and atherosclerotic plaque growth at the aortic roots. Stressed mice had increased levels of plasma DPP4 and decreased levels of plasma GLP-1 and adiponectin (APN) and adipose APN expression. Stress increased plaque macrophage infiltration, neovessel density, and elastin fragmentation, lessened the plaque collagen content, and increased the levels of toll-like receptor-2 (TLR2), TLR4, C-X-C chemokine receptor-4, cathepsins S and K, osteopontin, peroxisome proliferator-activated receptor-α, p16 INK4A , p21, and gp91phox mRNAs and/or proteins. Stressed aortas had also increased matrix metalloproteinase-2 (MMP-2) and MMP-9 activities. DPP4 inhibition with anagliptin reversed stress-related atherosclerotic lesion formation, and this benefit was abrogated by APN blocking. In vitro, the GLP-1 receptor agonist exenatide stimulated APN expression in 3T3-L1 cells. Conclusions These results indicate that the DPP4 inhibition-mediated benefits are likely attributable, at least in part, to attenuation of plaque inflammation, oxidative stress and proteolysis associated with GLP-1-mediated APN production in ApoE −/− mice under stress. Thus, DPP4 will be a novel therapeutic target for the treatment of stress-related cardiovascular disease.

Adrenal cortex and medulla are derived from mesoderm and ectoderm, respectively. Mixed corticomedullary tumors (MCMTs), comprising an intimately admixed population of both adrenal cortical cells and pheochromocytes in a single adrenal tumor, are extremely rare and its pathogenesis has remained unknown. Here, we report a case of MCMT whose cells co-expressed cortical and medullary antigens in the same tumor cells.[Case description]A 40-year-old woman was referred to our hospital for investigating Takotsubo cardiomyopathy following resection of uterine fibroids. An abdominal CT scan depicted a 24 mm tumor on her left adrenal gland. Her basal serum ACTH, cortisol levels and urinary cortisol were 13.8 pg/mL, 9.5 μg/dL, and 26.5 μg/day respectively. The cortisol level was normally suppressed by an administration of 1 mg dexamethasone (1.4 μg/dL). Plasma renin activity, aldosterone levels and urinary aldosterone were 15.0 ng/mL/h, 122 pg/mL, and 5.0 μg/day, respectively (with administration history of azosemide). On the other hand, her plasma adrenaline and noradrenaline levels were elevated as high as 177 pg/mL and 536 pg/mL, and urinary metanephrine and normetanephrine were 2.12 mg/day and 1.10 mg/day. A 123I-metaiodobenzylguanidine scan revealed high uptake in the tumor. After adequate adrenergic α-receptor blockage, left adrenalectomy was performed. Her postoperative endocrine and clinical findings were normalized without any further complications.[Pathology] Immunohistochemistry (IHC) revealed the presence of MCMT. Cells morphologically consistent with pheochromocytoma and adrenocortical cells were confirmed by immunostaining of chromogranin A and SF-1, respectively. Chromogranin A-positive medullary-derived and SF-1-positive cortical-derived tumor cells were intermixed in the chimeric fashion. In addition, some tumor cells were positive for both proteins, indicating hybrid nature of the cells. Tumor cells of cortical origin expressed CYP11β1, 3β-HSD, p450c21, and p450c17, but not CYP11β2. Non neoplastic adrenal cortex were atrophic, whereas the glomerulosa was hyperplastic positive for CYP11β2, consistent with diffuse hyperplasia and adrenal medullar unremarkable. [Conclusions:]The adrenal tumor was clinically diagnosed as pheochromocytoma, but the pathological findings did reveal cortisol production in the tumor and aldosterone overproduction in the accompanying cortex. This is the first case of MCMT co-expressing adrenal medullary and cortical antigens in the same tumor cells as hybrid cells.

Background The mechanism by which angiogenesis declines with aging remains largely unknown. Given that the plasma levels of adiponectin (APN) are decreased in the presence of ischemic cardiovascular disease, we explore the possible mechanisms by which APN/adiponectin receptor1 (AdipoR1) axis inactivation contributes to the decline in vascular regeneration capacity in elderly animals. Methods and results To study aging-related changes in the APN/AdipoR1 axis and its impact on ischemia-induced angiogenesis, a hindlimb ischemia model was applied to young and aged mice. Aging impaired ischemia-induced blood flow recovery. An ELISA showed that the aged mice had decreased plasma APN levels. Immunostaining showed lesser capillary formation in the aged mice. The aged ischemic muscles had decreased levels of AdipoR1, peroxisome proliferator activated receptor-γ (PPAR-γ), PPAR-γ co-activator 1α (PGC-1α), phospho-AMP-activated protein kinase α (p-AMPK-α), and B cell lymphoma-2 (Bcl-2) and increased levels of cleaved caspase-8 (C-caspase-8) and gp91phox/p22phox genes or/and proteins, nicotinamide adenine dinucleotide phosphate oxidase activity, superoxide production, and matrix metalloproteinase-2/−9 activity as well as increased numbers of infiltrated macrophages and leucocytes. In in vitro experiments, aged endothelial cells had negative changes in the levels of PPAR-γ, PGC-1α, p-AMPK-α, Bcl-2, and C-caspase-8 proteins in response to oxidative stress. Genetic interventions targeted toward APN and AdipoR1 negatively affected the targeted angiogenic protein levels in aged muscles and angiogenic actions and/or aged endothelial events. Conclusion These findings indicate that aging can reduce angiogenesis in response to hypoxia via an impaired APN-AdipoR1-dependent mechanism that may be mediated by PPAR-γ/PGC-1α signaling inactivation in advanced age.

β -Hydroxy- β -methylbutyrate (HMB) prevents deleterious muscle responses under pathological conditions, including tumor- and chronic steroid therapy–related muscle losses. Here, we investigated the hypothesis that HMB may modulate the balance between protein synthesis and degradation in the PI3K/Akt-mediated mammalian target of rapamycin (mTOR) and FoxO1/FoxO3a-dependent mechanisms in differentiated C2C12 muscle cells. We also tested the effect of HMB on the expression of MuRF-1 and atrogin-1 in response to the inflammatory stress. β -Hydroxy- β -methylbutyrate up-regulated phosphorylation of Akt and mTOR, and these effects were completely abolished in the presence of PI3K inhibitor LY294002. β -Hydroxy- β -methylbutyrate also up-regulated FoxO1 and FoxO3a phosphorylation, and these changes were inhibited by LY294002. Although, unexpectedly, HMB failed to reduce the expressions of atrophy-related atrogin-1 messenger RNA and the protein response to the proinflammatory cytokines tumor necrosis factor α plus interferon γ , HMB did attenuate the MuRF-1 expression. Thus, HMB appears to restore the balance between intracellular protein synthesis and proteolysis, likely via activation of the PI3K/Akt-dependent mTOR and FoxO1/FoxO3a signaling pathway and the reduction of tumor necrosis factor α /interferon γ –induced MuRF-1 expression, thereby ameliorating aging-related muscle atrophy.

We examined the anti-allergic effect of strawberry extract on human peripheral blood mononuclear cells (PBMCs) and atopic dermatitis model mice NC/NgaTndCrlj. The addition of strawberry extract suppressed total IgE production in the cedar pollen antigen Cry j 1-stimulated PBMCs. Flow cytometric analysis showed that strawberry extract decreased the rate of CD3+CD4+ helper T cells by 17.3% and increased the rate of CD3+CD8+ cytotoxic T cells by 19.7% in PBMCs. Moreover, the extract inhibited the expression level of GATA3 that is the master regulator of type 2 helper T cells (Th2) in human primary pan T cells isolated from PBMCs. Oral administration of strawberry extract lowered dermatitis scores and serum IgE levels in mice. In addition, it also decreased the GATA3 expression level in mouse blood cells. These results revealed that strawberry extract suppressed the severity of atopic dermatitis through the down-regulation of serum IgE by inhibition of Th2 differentiation.

Background: Dipeptidyl peptidase-4 (DPP4) inhibition exhibits multiple pleotrophic effects. Hematopoietic stem cell (HSC) activation has been implicated in the pathogenesis of stress-related metabolic disorder and cardiovascular disease. Given that interaction between β3-adrenergic receptor (Adrβ3) signaling and the immune system may link stress and the initiation and progression of disorders, we investigated whether DPP4 regulates immune over-reactions in a chronic stress mouse model, focusing on HSC activation. Methods and Results: Male 8-week-old mice fed a normal diet underwent chronic stress were randomly assigned to one of three groups and administered vehicle or a low or high dose of the DPP4 inhibitor anagliptin. Control mice were left undisturbed. The stress increased the blood and brain DPP4 activity, the levels of plasma adrenaline and noradrenaline, and the bone marrow (BM) niche cell adrenergic receptor (Adrβ3) expression, and it decreased the levels of plasma glucagon-like peptide (GLP-1) as well as brain GLP-1 receptor (GLP-1R) and BM Cxcl12 expressions. These changes were reversed by DPP4 inhibition. The stress activated the BM sca-1 high c-Kit high CD48 low CD150 high HSC proliferation, giving rise to high levels of blood leukocytes and monocytes. These DPP4 inhibition-related benefits were mimicked by DPP4 depletion and by GLP-1R activation. Adrβ3 inhibition mitigated BM Cxcl12 expressions and HSC activation. Conclusions: DPP4 activity appears to regulate chronic stress-induced BM HSC activation and inflammatory cell production via an Adrβ3-CXCLl12-dependent mechanism that is mediated by the GLP-1-GLP-1R axis, suggesting that the inhibition of DPP4 or the stimulation of GLP-1R may have applications in the treatment of inflammatory diseases.

We purified and identified an IgE suppressor from the strawberry ‘Toyonoka’, based on the decrease of IgE production in in vitro immunization (IVI). Gel filtration experiment indicated that fractions in a 15–48 kDa range and 98% identical nucleotides and >99% identical amino acids, respectively. The purified strawberry GAPDH suppressed total IgE production in IVI in a dose-dependent manner. From these results, we identified GAPDH as IgE suppressor in the strawberry. Our study may be applicable to the development of new methods to relieve allergic conditions using GAPDH and the screening of other functional factors for human health.

A fructose-based cell culture is suitable for the process control of protein production because of slow sugar consumption rate and low lactate accumulation. The fructose transporter, GLUT5, mediates its incorporation into cells and is required for the fructose-based culture. In order to produce efficiently recombinant IgG by metabolic control and co-expression with GLUT5 in a fructose-based medium, an IgG and GLUT5 co-expression vector was constructed and transfected into the human myeloma derived cell line, SC-01MFP, which produced stably recombinant proteins. The cell proliferation in the fructose-based medium was improved by the GLUT5 gene transfection. The recombinant IgG production of the cells cultured in the fructose-based medium exhibited about two-fold increase of that in the glucose-based medium. Flow cytometoric analysis indicated that the GLUT5 protein expression level in cell surface was increased in the fructose-based medium. An exogenous but not endogenous GLUT5 transcription activator remarkably raised IgG productivity in the fructose-based medium when compared to that in the glucose-based medium, suggesting that exogenous GLUT5 expression may be involved in it. The GLUT5 co-expression system may be useful for efficient production of recombinant proteins by the fructose-based cell culture.

Supplemental Digital Content is available in the text., Objective— Cathepsin S (CatS) participates in atherogenesis through several putative mechanisms. The ability of cathepsins to modify histone tail is likely to contribute to stem cell development. Histone deacetylase 6 (HDAC6) is required in modulating the proliferation and migration of various types of cancer cells. Here, we investigated the cross talk between CatS and HADC6 in injury-related vascular repair in mice. Approach and Results— Ligation injury to the carotid artery in mice increased the CatS expression, and CatS-deficient mice showed reduced neointimal formation in injured arteries. CatS deficiency decreased the phosphorylation levels of p38 mitogen-activated protein kinase, Akt, and HDAC6 and toll-like receptor 2 expression in ligated arteries. The genetic or pharmacological inhibition of CatS also alleviated the increased phosphorylation of p38 mitogen-activated protein kinase, Akt, and HDAC6 induced by platelet-derived growth factor BB in cultured vascular smooth muscle cells (VSMCs), and p38 mitogen-activated protein kinase inhibition and Akt inhibition decreased the phospho-HDAC6 levels. Moreover, CatS inhibition caused decrease in the levels of the HDAC6 activity in VSMCs in response to platelet-derived growth factor BB. The HDAC6 inhibitor tubastatin A downregulated platelet-derived growth factor–induced VSMC proliferation and migration, whereas HDAC6 overexpression exerted the opposite effect. Tubastatin A also decreased the intimal VSMC proliferation and neointimal hyperplasia in response to injury. Toll-like receptor 2 silencing decreased the phosphorylation levels of p38 mitogen-activated protein kinase, Akt, and HDAC6 and VSMC migration and proliferation. Conclusions— This is the first report detailing cross-interaction between toll-like receptor 2–mediated CatS and HDAC6 during injury-related vascular repair. These data suggest that CatS/HDAC6 could be a potential therapeutic target for the control of vascular diseases that are involved in neointimal lesion formation.

The mechanism by which angiogenesis declines with aging is not fully understood. Soluble vascular endothelial growth factor receptor 1 (VEGFR1) form (sFlt1) contributes to endothelial dysfunction in pathological conditions. However, the roles of sFlt1 in ischemia-induced neovascularizationof aged animals have not been investigated. To study aging-related sFlt1 change and its impact on ischemia-induced neovascularization, a hindlimb ischemia model was applied to young and aged mice. Blood flow imaging assay revealed that the blood flow recovery remained impaired throughout the follow-up period. At day 14, immunostaining showed lesser capillary formation in the aged mice. An ELISA showed that the aged mice had increased plasma sFlt-1 levels at indicated time points after surgery. On operative day 4, the aged ischemic muscles had decreased levels of p-VEGFR2 and p-Akt and increased levels of sFlt-1, Wnt5a, and SC35 genes or/and protein as well as increased numbers of inflammatory cells (macrophages and leucocytes) and matrix metalloproteinase-9 activity. Immnunofluorescence showed that Flt-1 was co-localized with CD11b+ macrophages of aged ischemic muscles. Hypoxia stimulated sFlt1 expression in CD11b+ cells of aged bone-marrow (BM), and this effect was diminished by siWnt5a. The cultured medium of aged mice BM-derived CD11b+ cells suppressed human endothelial cell (EC) and endothelial progenitor cell (EPC) angiogenic actions induced by VEGF, and these decreases were improved by treatment with siWnt5a-conditioned medium. Thus, aging appears to decline neovascularization in response to ischemic stress via the VEGFR2/Akt signaling inactivation in ECs and ECPs that is mediated by Wnt5a/SC35 axis activated macrophages-derived sFlt1 production in advanced age.

Introduction: The mineralocorticoid receptor has been implicated in the pathogenesis of chronic cardiorenal disease. 3-hydroxy-3methylgutaryl coenzyme A (HMG-CoA) reductase inhibitors, statins, improved renal dysfunction in patients with proteinuric kidney disease. Objective: We aimed to clarify the potential effect of a statin to enhance the inhibitory effect of an angiotensin type1 receptor blocker (ARB) on renal injury and the cellular mechanism of the effect of ARB on renal remodeling and proteinuria. Methods and Results: Dahl salt-sensitive (DS) rats on a high-salt diet were randomly assigned to four groups (n = 10 for each group) that were treated with either vehicle (0.5% carboxymethyl cellulose), a low or high dosage of olmesartan (1 or 3 mg/kg/d), or pitavastatin (1 mg/kg/d) plus olmesartan (1 mg/kg/d) from 12 to 19 weeks of age. Rats fed a low-salt diet served as age-matched controls. Rats on the high-salt diet developed massive proteinuria and glomerulosclerosis, and these changes were attenuated by olmesartan in a dose-dependent manner. The amounts of mRNAs for AT1R, mineralocorticoid receptor (MR), osteopontin, monocyte chemoattractant protein-1 and collagen type I, and the activities for matrix metalloproteinase-9 and cathepsin S were significantly higher in the failing kidneys of vehicle-treated rats than in the age-matched control rats; olmesartan significantly attenuated these changes. Olmesartan attenuated both the decrease in the ratio of reduced glutathione to oxidized glutathione and the increase in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity apparent in the kidney cortex of vehicle-treated rats. Furthermore, olmesartan inhibited kidney cortex vascular inflammation and renal fibrosis. The addition of pitavastatin significantly enhanced these beneficial effects by AT1R antagonism via anti-inflammation and anti-proteolysis. Conclusions: The beneficial renal effects of AT1R antagonism are likely attributable, at least in part, to the attenuation of renal oxidative stress and renal vascular inflammation induced by the AT1R-MR signaling interaction. The combination of statin with ARB may be a potential therapeutic strategy for renal injury with proteinuria.

Cathepsin K (CatK) is one of the most potent mammalian collagenases. We showed previously the increased expression of CatK in human and animal atherosclerotic lesions. Here, we hypothesized that ablation of CatK mitigates injury-induced neointimal hyperplasia. Male wild-type (CatK +/+ ) and CatK-deficient (CatK −/− ) mice underwent ligation or a combination of ligation and polyethylene cuff-replacement injuries to the right common carotid artery just proximal to its bifurcation, and they were then processed for morphological and biochemical studies at specific time points. On operative day 28, CatK −/− significantly reduced neointimal formation and neovessel formation in both single- and combination-injured arteries compared with the Cat K +/+ mice. At early time points, CatK −/− reduced the lesion macrophage contents and medial smooth muscle cell proliferation, the mRNA levels of monocyte chemoattractant protein-1, toll-like receptor-2, toll-like receptor-4, chemokine ligand-12, and the gelatinolytic activity related to matrix metalloproteinase-2/-9. An aorta-explant assay revealed that smooth muscle cell movement was impaired in the CatK −/− mice compared with the CatK +/+ mice. In addition, the smooth muscle cells and macrophages from CatK −/− mice had less invasive ability through a reconstituted basement membrane barrier. This vasculoprotective effect was mimicked by Cat inhibition with trans -epoxysuccinyl-L-leucylamido-{4-guanidino} butane (E64 d ). These results demonstrate an essential role of CatK in neointimal lesion formation in response to injury, possibly via the reduction of toll-like receptor-2/-4–mediated inflammation and smooth muscle cell proliferation, suggesting a novel therapeutic strategy for the control of endovascular treatment–related restenosis by regulating CatK activity.

A fructose containing cell culture medium has the advantage of low lactate production and a small pH change, leading to cell and product stability. But, not all cell lines grow well in the medium, and the fructose transporter, GLUT5, is related to it [1]. Thus, we developed an efficient production system of recombinant proteins by metabolic control and co-expression with GLUT5 in a fructose-based medium [2]. In this report, the availability of the GLUT5 co-expression system was indicated in CHO-K1 and the human cell line, SC-01MFP [3]. As a model, an IgG and GLUT5 co-expression vector was constructed and transfected into cells. When the transfected CHO-K1 and SC-01MFP cells were cultured in the fructose-based medium, both IgG productions were increased up to about two-fold of that cultured in the glucose-based medium (Table ​(Table1).1). Our study may be useful for efficient production of recombinant proteins using the fructose-based cell culture. In particular, the production in SC-01MFP cells is valuable for functional analysis of recombinant proteins with a human glycosylation profile. Table 1 Proliferation and IgG production in the fructose-based medium.

The interactions between the renin-angiotensin system and neovascularization in atherosclerotic plaque development are unclear. We investigated the effects of angiotensin II type 1 receptor antagonism in the pathogenesis of atherosclerosis in apolipoprotein E–deficient (ApoE −/− ) mice with a special focus on plaque neovascularization. ApoE −/− mice fed a high-fat diet were randomly assigned to 1 of 2 groups and administered vehicle or olmesartan for 12 weeks. Quantification of plaque areas at the aortic root and in the thoracic and abdominal aorta revealed that, in all 3 of the regions, olmesartan reduced intimal neovessel density and the mRNA levels of toll-like receptor (TLR) 2 and TLR4. Olmesartan increased the levels of collagen and elastin, reduced the level of macrophages in the aortic root, and reduced the mRNA and the activity of matrix metalloproteinase (MMP) 2 in aortic roots and thoracic aortas. Aortic ring assay revealed that olmesartan-treated ApoE −/− mice had a markedly lower angiogenic response than that of untreated ApoE −/− mice. Bone marrow–derived endothelial progenitor cell-like c-Kit + cells from olmesartan-treated ApoE −/− mice showed marked impairment of cellular functions and lower expression of TLR2/TLR4 and MMP-2 compared with those of untreated controls. MMP-2 deficiency reduced intimal neovessel density and atherosclerotic lesion formation. Olmesartan and small-interfering RNA targeting TLR2 reduced the levels of TLR2, and MMP-2 mRNA induced angiotensin II in cultured endothelial cells. Angiotensin II type 1 receptor antagonism appears to inhibit intimal neovascularization in ApoE −/− mice, partly by reducing TLR2/TLR4-mediated inflammatory action and MMP activation, thus decreasing atherosclerotic plaque growth and increasing plaque instability.

We examined the anti-allergic action of strawberry extracts in pollinosis by using an in vitro IgE inducing method. It was found that several kinds of the strawberries suppressed total IgE production as much as epicatechin gallate that is well known as an anti-allergic factor. The IgE decrease was correlated to the decrease of SOCS3 specifically expressed in Th2 cells that promote IgE production. In addition, Biacore analysis revealed that there is a factor with the ability to bind with the ceder pollen allergen Cryj1 in the strawberry extracts. These results suggest that the strawberry extracts may suppress the allergic reaction by acting on both Th2 cells and Cryj1 allergen.

IgA is thought to be critical for intestinal immune system. Generally, the effect of food components on intestinal immune system is evaluated by animal experiments. However, the development of simple screening method using human cell culture makes them more cost effective and high-throughput. Thus, we investigated the culture condition of the IgA-producing human myeloma cell line KHM-1B that has unstable culture properties such as cluster formation, resulting in rapid cell death. Considering high-throughput screening, the culture condition was examined in 96-well culture plates and the following parameters were periodically measured: 1. Time to form clusters, 2. Viability, and 3. IgA production. As a result, we obtained a clone which could grow well and produce IgA antibodies stably. The doubling time of this clone was about 72 h, and continuously generated IgA antibodies for 2 months. Then, in order to examine the effect of food components on IgA production using this culture system, various components such as ascorbic acid, raffinose, lactoferrin, fructooligosaccharides, casein tryptic digests, strawberry crude extracts, and IL-6 as a positive control were added to the KHM-1B cell culture medium. The extracts from several kinds of strawberry as well as IL-6 were found to promote IgA production in KHM-1B cells. These results might be useful for screening of food derived IgA-promoting factors.

We found that the increased expression of fructose transporter, GLUT5 improved cell proliferation and antibody production in human hybridoma and myeloma cell lines cultured in the fructose-based medium. The IgG and GLUT5 expression vectors cotransfected myeloma clones showed a tendency to increase IgG production in the fructose-based medium more than in the glucose-based medium. The both IgG and GLUT5 expression vector transfected myeloma cells increased its tendency, suggesting that IgG expression might be activated with GLUT5 expression in the fructose-based medium when IgG gene was located in the vicinity of GLUT5 gene.

Neutrophil elastase (NE), a serine protease released from the azurophil granules of activated neutrophil, proteolytically cleaves multiple cytokines, and cell surface proteins. In the present study, we examined whether NE affects the biological abilities of angiogenic growth factors such as basic-fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). NE degraded bFGF and VEGF in a time- and concentration-dependent manner, and these degradations were suppressed by sivelestat, a synthetic inhibitor of NE. The bFGF- or VEGF-mediated proliferative activity of human umbilical vein endothelial cells was inhibited by NE, and the activity was recovered by sivelestat. Furthermore, NE reduced the bFGF- or VEGF-induced tubulogenic response of the mice aortas, ex vivo angiogenesis assay, and these effects were also recovered by sivelestat. Neutrophil-derived NE degraded potent angiogenic factors, resulting in loss of their angiogenic activity. These findings provide additional insight into the role played by neutrophils in the angiogenesis process at sites of inflammation.