Piezo type mechanosensitive ion channel component 1 functions as a regulator of the cell fate determination of mesenchymal stem cells.

The extracellular environment regulates the dynamic behaviors of cells. However, the effects of hydrostatic pressure (HP) on cell fate determination of mesenchymal stem cells (MSCs) are not clearly understood. Here, we established a cell culture chamber to control HP. Using this system, we found that the promotion of osteogenic differentiation by HP is depend on bone morphogenetic protein 2 (BMP2) expression regulated by Piezo type mechanosensitive ion channel component 1 (PIEZO1) in MSCs. The PIEZO1 was expressed and induced after HP loading in primary MSCs and MSC lines, UE7T-13 and SDP11. HP and Yoda1, an activator of PIEZO1, promoted BMP2 expression and osteoblast differentiation, whereas inhibits adipocyte differentiation. Conversely, PIEZO1 inhibition reduced osteoblast differentiation and BMP2 expression. Furthermore, Blocking of BMP2 function by noggin inhibits HP induced osteogenic maker genes expression. In addition, in an in vivo model of medaka with HP loading, HP promoted caudal fin ray development whereas inhibition of piezo1 using GsMTx4 suppressed its development. Thus, our results suggested that PIEZO1 is responsible for HP and could functions as a factor for cell fate determination of MSCs by regulating BMP2 expression.

Endoplasmic Reticulum Stress in Mice Increases Hepatic Expression of Genes Carrying a Premature Termination Codon via a Nutritional Status-Independent GRP78-Dependent Mechanism.

Nonsense-mediated mRNA decay (NMD) degrades mRNAs carrying a premature termination codon (PTC) in eukaryotes. Cellular stresses, including endoplasmic reticulum (ER) stress, inhibit NMD, and up-regulate PTC-containing mRNA (PTC-mRNA) levels in several cell lines. However, whether similar effects exist under in vivo conditions that involve systemic nutritional status is unclear. Here, we compared the effects of pharmacological induction of ER stress with those of nutritional interventions on hepatic PTC-mRNA levels in mice. In mouse livers, the ER stress inducer tunicamycin increased PTC-mRNA levels of endogenous marker genes. Tunicamycin decreased body weight and perturbed nutrient metabolism in mice. Food restriction or deprivation mimicked the effect of tunicamycin on weight loss and metabolism, but did not increase PTC-mRNA levels. Hyperphagia-induced obesity also had little effect on hepatic PTC-mRNA levels. Meanwhile, in mouse liver phosphorylation of eIF2α, a factor that regulates NMD, was increased by both tunicamycin and nutritional interventions. Hepatic expression of GRP78, a central chaperone in ER stress responses, was increased by tunicamycin but not by the nutritional interventions. In cultured liver cells (Hepa), exogenous overexpression of a phosphomimetic eIF2α failed to increase PTC-mRNA levels. However, GRP78 overexpression in Hepa cells increased PTC-mRNA and PTC-mRNA-derived protein levels. ER stress promoted localization of GRP78 to mitochondria, and exogenous expression of a GRP78 fusion protein targeted to mitochondria mimicked the effect of wild type GRP78. These results indicate that GRP78, but not nutritional status, is a potent up-regulator of hepatic PTC-mRNA levels during induction of ER stress in vivo. J. Cell. Biochem. 118: 3810-3824, 2017. © 2017 Wiley Periodicals, Inc.

Mechanisms Underlying Activation of α1-Adrenergic Receptor-Induced Trafficking of AQP5 in Rat Parotid Acinar Cells under Isotonic or Hypotonic Conditions.

Defective cellular trafficking of aquaporin-5 (AQP5) to the apical plasma membrane (APM) in salivary glands is associated with the loss of salivary fluid secretion. To examine mechanisms of α1-adrenoceptor (AR)-induced trafficking of AQP5, immunoconfocal microscopy and Western blot analysis were used to analyze AQP5 localization in parotid tissues stimulated with phenylephrine under different osmolality. Phenylephrine-induced trafficking of AQP5 to the APM and lateral plasma membrane (LPM) was mediated via the α1A-AR subtype, but not the α1B- and α1D-AR subtypes. Phenylephrine-induced trafficking of AQP5 was inhibited by ODQ and KT5823, inhibitors of nitric oxide (NO)-stimulated guanylcyclase (GC) and protein kinase (PK) G, respectively, indicating the involvement of the NO/ soluble (c) GC/PKG signaling pathway. Under isotonic conditions, phenylephrine-induced trafficking was inhibited by La(3+), implying the participation of store-operated Ca(2+) channel. Under hypotonic conditions, phenylephrine-induced trafficking of AQP5 to the APM was higher than that under isotonic conditions. Under non-stimulated conditions, hypotonicity-induced trafficking of AQP5 to the APM was inhibited by ruthenium red and La(3+), suggesting the involvement of extracellular Ca(2+) entry. Thus, α1A-AR activation induced the trafficking of AQP5 to the APM and LPM via the Ca(2+)/ cyclic guanosine monophosphate (cGMP)/PKG signaling pathway, which is associated with store-operated Ca(2+) entry.

C-terminal region of GADD34 regulates eIF2α dephosphorylation and cell proliferation in CHO-K1 cells.

GADD34 is a member of a growth arrest and DNA damage (GADD)-inducible gene family. Here, we established a novel Chinese hamster ovary (CHO)-K1-derived cell line, CHO-K1-G34M, which carries a nonsense mutation (termed the Q525X mutation) in the GADD34 gene. The Q525X mutant protein lacks the C-terminal 66 amino acids required for GADD34 to bind to and activate protein phosphatase 1 (PP1). We investigated the effects of GADD34 with or without the Q525X mutation on the phosphorylation status of PP1 target proteins, including the α subunit of eukaryotic initiation factor 2 (eIF2α) and glycogen synthase kinase 3ß (GSK3ß). CHO-K1-G34M cells had higher levels of eIF2α phosphorylation compared to the control CHO-K1-normal cells both in the presence and absence of endoplasmic reticulum stress. Overexpression of the wild-type GADD34 protein in CHO-K1-normal cells largely reduced eIF2α phosphorylation, while overexpression of the Q525X mutant did not produce similar reductions. Meanwhile, neither wild type nor Q525X mutation of GADD34 affected the GSK3ß phosphorylation status. GADD34 also did not affect the canonical Wnt signaling pathway downstream of GSK3ß. Cell proliferation rates were higher, while expression levels of the cyclin-dependent kinase inhibitor p21 were lower in CHO-K1-G34M cells compared to the CHO-K1-normal cells. The GADD34 Q525X mutant had a reduced ability to inhibit cell proliferation and enhance p21 expression of the CHO-K1-normal cells compared to the wild-type GADD34 protein. These results suggest that the GADD34 protein C-terminal plays important roles in regulating not only eIF2α dephosphorylation but also cell proliferation in CHO-K1 cells.

Activation of muscarinic receptors in rat parotid acinar cells induces AQP5 trafficking to nuclei and apical plasma membrane.

BACKGROUND: The subcellular distribution of aquaporin-5 (AQP5) in rat parotid acinar cells in response to muscarinic acetylcholine receptor (mAChR) activation remains unclear. METHODS: Immunoconfocal and immunoelectron microscopy were used to visualize the distribution of AQP5 in parotid acinar cells. Western blotting was used to analyze AQP5 levels in membranes. To clarify the characteristics of membrane domains associated with AQP5, detergent solubility and sucrose-density flotation experiments were performed. RESULTS: Under control conditions, AQP5 was diffusely distributed on the apical plasma membrane (APM) and apical plasmalemmal region and throughout the cytoplasm. Upon mAChR activation, AQP5 was predominantly located in the nucleus, APM and lateral plasma membrane (LPM). Subsequently, localization of AQP5 in the nucleus, APM and LPM was decreased. Prolonged atropine treatment inhibited mAChR agonist-induced translocation of AQP5 to the nucleus, APM and LPM. AQP5 levels were enhanced in isolated nuclei and nuclear membranes prepared from parotid tissues incubated with mAChR agonist. mAChR agonist induced AQP5 levels in both soluble and insoluble nuclear fractions solubilized with Triton X-100 or Lubrol WX. Small amounts of AQP5 in nuclei were detected using low-density sucrose gradient. When AQP5 was present in the nuclear membrane, nuclear size decreased. CONCLUSION: The activation of mAChR induced AQP5 translocation to the nucleus, APM and LPM, and AQP5 may trigger water transport across the nuclear membrane and plasma membrane in rat parotid acinar cells. GENERAL SIGNIFICANCE: AQP5 translocates to the nuclear membrane and may trigger the movement of water, inducing shrinkage of the nucleus and the start of nuclear functions.

Membrane topology of murine glycerol-3-phosphate acyltransferase 2.

Glycerol-3-phosphate acyltransferase (GPAT) is a rate-limiting enzyme in mammalian triacylglycerol biosynthesis. GPAT is a target for the treatment of metabolic disorders associated with high lipid accumulation. Although the molecular basis for GPAT1 activation has been investigated extensively, the activation of other isoforms, such as GPAT2, is less well understood. Here the membrane topology of the GPAT2 protein was examined using an epitope-tag-based method. Exogenously expressed GPAT2 protein was present in the membrane fraction of transformed HEK293 cells even in the presence of Na(2)CO(3) (100 mM), indicating that GPAT2 is a membrane-bound protein. Trypsin treatment of the membrane fraction degraded the N-terminal (FLAG) and C-terminal (myc-epitope) protein tags of the GPAT2 protein. Bioinformatic analysis of the GPAT2 protein sequence indicated four hydrophobic sequences as potential membrane-spanning regions (TM1-TM4). Immunoblotting of the myc-epitope tag, which was inserted between each TM region of the GPAT2 protein, showed that the amino acid sequence between TM3 and TM4 was protected from trypsin digestion. These results suggest that the GPAT2 protein has two transmembrane segments and that the N-terminal and C-terminal regions of this protein face the cytoplasm. These results also suggest that the enzymatically active motifs I-III of the GPAT2 protein face the cytosol, while motif IV is within the membrane. It is expected that the use of this topological model of GPAT2 will be essential in efforts to elucidate the molecular mechanisms of GPAT2 activity in mammalian cells.

Interaction between PKR and PACT mediated by LPS-inducible NF-κB in human gingival cells.

The double-stranded RNA-dependent protein kinase (PKR) is a serine/threonine kinase expressed constitutively in mammalian cells. PKR is activated upon virus infection by double-stranded RNA (dsRNA), and plays a critical role in host antiviral defense mechanisms. PKR is also known to regulate various biological responses, including cell differentiation and apoptosis. However, whether PKR is involved in the progress of periodontitis is not clear. The present study explained the phosphorylation of PKR by LPS in the human gingival cell line, Sa3. Expression of genes encoding LPS receptors was detected in Sa3 cells and treatment of cells with 1 µg/mL LPS for 6 h caused PKR phosphorylation. LPS elevated the expression of the protein activator of PKR (PACT) mRNA and protein, followed by the enhanced association between PACT and PKR within 3 h. In addition, LPS treatment induced the translocation of NF-κB to the nucleus after 30 min, and inhibition of NF-κB decreased the PACT-PKR interaction induced by LPS. The level of pro-inflammatory cytokine mRNA, including interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFα), appeared within 45 min and reached at the maximal levels by 90 min after the addition of LPS. This induction of pro-inflammatory cytokines was not affected by RNAi-mediated silencing of PKR and a pharmacological inhibitor of PKR, whereas the inhibition of NF-κB decreased it. These results indicated that LPS induces PKR phosphorylation and the PACT-PKR association in Sa3 cells. Our results also suggest that NF-κB is involved in the PACT-PKR interaction and the production of pro-inflammatory cytokines in periodontitis.

Role of the O-linked ß-N-acetylglucosamine in the cardioprotection induced by isoflurane.

BACKGROUND: Cardiac protection by volatile anesthetic-induced preconditioning and ischemic preconditioning have similar signaling pathways. Recently, it was reported that augmentation of protein modified with O-linked ß-N-acetylglucosamine (O-GlcNAc) contributes to cardiac protection. This study investigated the role of O-GlcNAc in cardiac protection induced by anesthetic-induced preconditioning. METHODS: O-GlcNAc-modified proteins were visualized by immunoblotting. Tolerance against ischemia or reperfusion was tested in vivo (n = 8) and in vitro (n = 6). The opening of the mitochondrial permeability transition pore (mPTP) upon oxidative stress was examined in myocytes treated with calcein AM (n = 5). Coimmunoprecipitation and enzymatic labeling were performed to detect the mitochondrial protein responsible for the mPTP opening. RESULTS: Isoflurane treatment and the consequent augmentation of O-GlcNAc concentrations reduced the infarct size (26 ± 5% [mean ± SD], P < 0.001) compared with the control. The protective effect of O-GlcNAc was eliminated in the group pretreated with the O-GlcNAc transferase inhibitor alloxan (39 ± 5%, P < 0.001). Myocyte survival also showed the same result in vitro. Formation of the mPTP was abrogated in the isoflurane-treated cells (86 ± 4%, P < 0.001) compared with the control and alloxan-plus-isoflurane-treated cells (57 ± 7%, P < 0.001). Coimmunoprecipitation and enzymatic labeling studies revealed that the O-GlcNAc-modified, voltage-dependent anion channel restained the mPTP opening. CONCLUSIONS: Isoflurane induced O-GlcNAc modification of mitochondrial voltage-dependent anion channel. This modification inhibited the opening of the mPTP and conferred resistance to ischemia-reperfusion stress.

Effects of a 1.5 T time-varying magnetic field on cell volume regulation of bovine adrenal chromaffin cells in hyposmotic media.

Effects of a time-varying magnetic field on cell volume regulation by hyposmotic stress in cultured bovine adrenal chromaffin cells were examined. Through regulatory volume decrease (RVD), cell volume of chromaffin cells that were incubated in a hypotonic medium initially increased, reached a peak and finally recovered to the initial value. Two hour exposure to a magnetic field and addition of cytochalasin D increased peak value and delayed return to initial value. Intracellular F-actin contents initially decreased but returned to normal levels after 10 sec. Two hour exposure to the magnetic field and addition of cytochalasin D continuously reduced the F-actin content. Results suggest that exposure to the magnetic field stimulated disruption of the actin cytoskeleton and that the disruption delayed the recovery to the volume prior to osmotic stress.

Abnormal subcellular localization of AQP5 and downregulated AQP5 protein in parotid glands of streptozotocin-induced diabetic rats.

BACKGROUND: The mechanisms underlying diabetic xerostomia have not been clarified in relation with aquaporin-5 (AQP5) subcellular localization in salivary glands. METHODS: Western blotting, real-time PCR, and immunocytochemistry were used to analyse AQP5 protein levels and mRNA expression. AQP5 protein levels were measured in the apical plasma membrane (APM) and detergent-insoluble fraction prepared from streptozotocin-diabetic rat parotid glands. RESULTS: Despite an increase in AQP5 mRNA, AQP5 protein levels were decreased in diabetic parotid glands compared with controls. Immunohistochemical studies indicated that AQP5, under unstimulated conditions, colocalised with flotillin-2 and GM1 with a diffuse pattern in the apical cytoplasm of acinar and duct cells in both control and diabetic rats. Ten minutes after intravenous injection of muscarinic agonist cevimeline, AQP5 was dramatically increased together with flotillin-2 and GM1 in the APM of parotid acinar and duct cells of control but not diabetic rats. Sixty minutes after injection, AQP5 was located in a diffuse pattern in the apical cytoplasm in both rats. Treatment of the parotid tissues with cevimeline for 10min increased the Triton X-100 solubility of AQP5 in control but not diabetic rats. Administration of insulin to diabetic rats tended to restore the cevimeline-induced translocation of AQP5. CONCLUSION: Lack of AQP5 translocation in the salivary gland in response to a muscarinic agonist and downregulation of AQP5 protein might lead to diabetic xerostomia. GENERAL SIGNIFICANCE: Cevimeline is useful to cure diabetic xerostomia under insulin administration.

Intermedilysin induces EGR-1 expression through calcineurin/NFAT pathway in human cholangiocellular carcinoma cells.

Intermedilysin (ILY) is a cholesterol-dependent cytolysin produced by Streptococcus intermedius, which is associated with human brain and liver abscesses. Although intrahepatic bile duct cells play a valuable role in the pathogenesis of liver abscess, the molecular mechanism of ILY-treated intrahepatic bile duct cells remains unknown. In this study, we report that ILY induced a nuclear accumulation of intracellular calcium ([Ca(2+)]i) in human cholangiocellular cells HuCCT1. We also demonstrate that 10 ng/ml ILY induced NFAT1 dephosphorylation and its nuclear translocation in HuCCT1 cells. In contrast to the result that ILY induced NF-κB translocation in human hepatic HepG2 cells, ILY did not affect NF-κB localization in HuCCT1 cells. Dephosphorylation and nuclear translocation of NFAT1 caused by ILY were prevented by [Ca(2+)]i calcium chelator, BAPTA/AM, and calcineurin inhibitors, cyclosporine A and tacrolimus. ILY induced early growth response-1 (EGR-1) expression and it was inhibited by the pre-treatment with cyclosporine A, indicating that the calcineurin/NFAT pathway was involved in EGR-1 expression in response to ILY. ILY-induced calcineurin/NFAT1 activation and sequential EGR-1 expression might be related to the pathogenesis of S. intermedius in human bile duct cells.

Effects of exposure to a time-varying 1.5 T magnetic field on the neurotransmitter-activated increase in intracellular Ca(2+) in relation to actin fiber and mitochondrial functions in bovine adrenal chromaffin cells.

BACKGROUND: It has been reported that exposure to electromagnetic fields influences intracellular signal transduction. We studied the effects of exposure to a time-varying 1.5 T magnetic field on membrane properties, membrane cation transport and intracellular Ca(2+) mobilization in relation to signals. We also studied the mechanism of the effect of exposure to the magnetic field on intracellular Ca(2+) release from Ca(2+) stores in adrenal chromaffin cells. METHODS: We measured the physiological functions of ER, actin protein, and mitochondria with respect to a neurotransmitter-induced increase in Ca(2+) in chromaffin cells exposed to the time-varying 1.5 T magnetic field for 2h. RESULTS: Exposure to the magnetic field significantly reduced the increase in [Ca(2+)]i. The exposure depolarized the mitochondria membrane and lowered oxygen uptake, but did not reduce the intracellular ATP content. Magnetic field-exposure caused a morphological change in intracellular F-actin. F-actin in exposed cells seemed to be less dense than in control cells, but the decrease was smaller than that in cytochalasin D-treated cells. The increase in G-actin (i.e., the decrease in F-actin) due to exposure was recovered by jasplakinolide, but inhibition of Ca(2+) release by the exposure was unaffected. CONCLUSIONS AND GENERAL SIGNIFICANCE: These results suggest that the magnetic field-exposure influenced both the ER and mitochondria, but the inhibition of Ca(2+) release from ER was not due to mitochondria inhibition. The effect of eddy currents induced in the culture medium may indirectly influence intracellular actin and suppress the transient increase in [Ca(2+)]i.

Effect of angiotensin II on iron-transporting protein expression and subsequent intracellular labile iron concentration in human glomerular endothelial cells.

Angiotensin II (Ang II)-induced endothelial injury, which is associated with atherosclerosis, is believed to be mediated by intracellular reactive oxygen species (ROS) through stimulation of nicotinamide adenine dinucleotide phosphate oxidase (NOX). Iron is essential for the amplification of oxidative stress. In this study, we investigated whether Ang II altered iron metabolism and whether the Ang II-induced endothelial injury is attributable to changes in iron metabolism of human glomerular endothelial cells (HGECs). When 90% iron-saturated human transferrin (90% Tf) was applied to HGECs without Ang II, the labile ferrous iron level was same as the effect of control in spite of a significant increase in the total cellular iron concentration. Treatment with Ang II and 30% Tf or 90% Tf significantly (P<0.01) increased the intracellular iron concentration, as well as labile ferrous iron and protein oxidation levels, compared with the effect of separate administration of each compound. Ang II treatment facilitated the protein expression of the Tf receptor, divalent metal transporter 1, and ferroportin 1 in a dose- and time-dependent manner. It was also found that simultaneous exposure of HGECs to Ang II and 90% Tf accelerated hydroxyl radical production, as shown by using an electron paramagnetic resonance spectrometer. These results suggest that Ang II not only induces production of ROS by NOX activation but also iron incorporation followed by an increase in labile iron in HGECs. Both of these events may participate in the progression of oxidative stress because of endothelial cell dysfunction through ferrous iron-mediated ROS generation.

Identification of aquaporin-5 and lipid rafts in human resting saliva and their release into cevimeline-stimulated saliva.

BACKGROUND: It is unknown whether AQP5 and lipid rafts are released into human unstimulated (resting) saliva and saliva in response to secretagogues. METHODS: In order to quantitate the salivary concentration of AQP5, we produced a polyclonal antibody for human AQP5 and developed an enzyme-like immunosorbent assay (ELISA). RESULTS: AQP5 and lipid rafts were identified in human resting saliva. The amount of AQP5 in resting saliva showed a diurnal variation with high levels during waking hours, and an age-related decrease in AQP5 was coincident with the volume of resting saliva. Cevimeline, a muscarinic acetylcholine receptor (mAChR) agonist, induced the release of AQP5 with lipid rafts, amylase, mucin, and lysozyme. Changes in saliva AQP5 levels after cevimeline administration occurred simultaneously with changes in saliva flow rates. Confocal microscopy revealed that AQP5 was located in the apical plasma membrane and showed a diffuse pattern in parotid glands under resting conditions. Following cevimeline administration, AQP5 was predominantly associated with the APM and was localized in the lumen. GENERAL SIGNIFICANCE: AQP5 and lipid rafts were released with salivary proteins from human salivary glands by the stimulation of M3 mAChRs, and that changes in saliva AQP5 levels can be used as an indicator of salivary flow rate and also as a useful index of M3 mAChR agonist's action on human salivary glands.

Reducing effect of feeding powdered nacre of Pinctada maxima on the visceral fat of rats.

An abdominal fat accumulation complicated by high blood triglycerides is regarded as a risk factor of metabolic syndrome. Feeding powdered nacre, mother of pearl, from Pinctada maxima, resulted in reduced body weight, visceral fat amount, and blood triglyceride level without influencing the food intake, body length, or amount of muscular tissue, suggesting that nacre powder specifically could decrease visceral fat.

Effects of estradiol and progesterone on the proinflammatory cytokine production by mononuclear cells from patients with chronic hepatitis C.

AIM: To investigate the effects of estradiol (E2) and progesterone on the unstimulated and oxidative stress-stimulated production of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-8, and macrophage chemotactic protein (MCP)-1 by peripheral blood mononuclear cells (PBMCs) from patients with chronic hepatitis C and healthy controls. METHODS: The PBMCs were separated from age-matched 72 males and 71 females with and without chronic hepatitis C, who were divided into two groups based on a mean menopausal age of 50 years. Oxidative stress was induced by hydrogen peroxide in the cells incubated in serum-free media. Cytokines in the culture supernatant were measured by an enzyme-linked immunosorbent assay. RESULTS: The highest levels of the spontaneous production of TNF-alpha, IL-1beta, IL-8, and MCP-1 by the unstimulated PBMCs were in the older male patients with chronic hepatitis C and the lowest levels were in the pre-menopausal female healthy controls. E2 inhibited the cytokine production by the unstimulated PBMCs from the older male and post-menopausal female patients, which was further stimulated by progesterone. The exposure to hydrogen peroxide in the PBMCs from the younger male and pre-menopausal female healthy subjects induced the production of cytokines. The change rates of the hydrogen peroxide-stimulated cytokine production were suppressed by E2 and enhanced by progesterone. CONCLUSION: These findings suggest that E2 may play a favorable role in the course of persistent liver injury by preventing the accumulation of monocytes-macrophages and by inhibiting proinflammatory cytokine production, whereas progesterone may counteract the favorable E2 effects.

Opposing effects of estradiol and progesterone on the oxidative stress-induced production of chemokine and proinflammatory cytokines in murine peritoneal macrophages.

In inflammatory and oxidative liver injury, virus proteins and reactive oxygen species are involved in the regulation of proinflammatory cytokine production by macrophages. This study investigated the effects of estradiol (E2) and progesterone on the unstimulated and oxidative stress-stimulated production of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, macrophage inflammatory protein (MIP)-2, and macrophage chemotactic protein (MCP)-1 by peritoneal macrophages isolated from male and female mice. E2 inhibited the cytokine production of TNF-alpha, IL-1beta, MIP-2, and MCP-1 by the unstimulated macrophages from males and females, which was then further stimulated by progesterone. The exposure to hydrogen peroxide in the macrophages from both sexes induced the production of cytokine. The hydrogen peroxide-stimulated cytokine production was suppressed by E2 and enhanced by progesterone. The sex hormone effects on the unstimulated and stimulated macrophages were blocked by their receptor antagonists and showed no significant difference between male and female subjects. These findings suggest that E2 may play a favorable role in the course of persistent liver injury, by inhibiting proinflammatory cytokine production, which, in addition, progesterone may counteract the favorable E2 effects through their receptors.

Subcutaneous injection, from birth, of epigallocatechin-3-gallate, a component of green tea, limits the onset of muscular dystrophy in mdx mice: a quantitative histological, immunohistochemical and electrophysiological study.

Dystrophic muscles suffer from enhanced oxidative stress. We have investigated whether administration of an antioxidant, epigallocatechin-3-gallate (EGCG), a component of green tea, reduces their oxidative stress and pathophysiology in mdx mice, a mild phenotype model of human Duchenne-type muscular dystrophy. EGCG (5 mg/kg body weight in saline) was injected subcutaneously 4x a week into the backs of C57 normal and dystrophin-deficient mdx mice for 8 weeks after birth. Saline was injected into normal and mdx controls. EGCG had almost no observable effects on normal mice or on the body weights of mdx mice. In contrast, it produced the following improvements in the blood chemistry, muscle histology, and electrophysiology of the treated mdx mice. First, the activities of serum creatine kinase were reduced to normal levels. Second, the numbers of fluorescent lipofuscin granules per unit volume of soleus and diaphragm muscles were significantly decreased by about 50% compared to the numbers in the corresponding saline-treated controls. Third, in sections of diaphragm and soleus muscles, the relative area occupied by histologically normal muscle fibres increased significantly 1.5- to 2-fold whereas the relative areas of connective tissue and necrotic muscle fibres were substantially reduced. Fourth, the times for the maximum tetanic force of soleus muscles to fall by a half increased to almost normal values. Fifth, the amount of utrophin in diaphragm muscles increased significantly by 17%, partially compensating for the lack of dystrophin expression.

Role of alpha1-acid glycoprotein in therapeutic antifibrotic effects of imatinib with macrolides in mice.

RATIONALE: Imatinib is an inhibitor of platelet-derived growth factor receptors. We have reported that treatment with imatinib inhibited bleomycin-induced pulmonary fibrosis in mice. However, late treatment with imatinib had no effect. OBJECTIVES: To clarify why imatinib had no antifibrotic effect when its administration was delayed, we focused on alpha(1)-acid glycoprotein (AGP), because it was reported to bind imatinib and mediate drug resistance. METHODS: The concentration of AGP in serum of mice and patients with idiopathic pulmonary fibrosis was measured by radial immunodiffusion testing. The effects of AGP in vitro were evaluated by assaying the growth of lung fibroblasts. We examined the combined effects of erythromycin (EM) or clarithromycin (CAM) on bleomycin-induced pulmonary fibrosis in mice. MEASUREMENTS AND MAIN RESULTS: Addition of AGP abrogated imatinib-mediated inhibition of the growth of fibroblasts. However, treatment with EM or CAM restored the growth-inhibitory effects of imatinib. The elevated level of AGP was detected in serum and lung homogenates in bleomycin-exposed mice and reached a plateau on Day 14. Imatinib alone did not ameliorate pulmonary fibrosis when treatment was started on Day 15, whereas coadministration of imatinib and EM or CAM significantly reduced the fibrogenesis via inhibition of the growth of fibroblasts in vivo. Serum levels of AGP were higher in patients with idiopathic pulmonary fibrosis than in healthy subjects. CONCLUSIONS: AGP is an important regulatory factor modulating the ability of imatinib to prevent pulmonary fibrosis in mice, and combined therapy with imatinib and EM or CAM might be useful for treatment of pulmonary fibrosis.

Endothelial injury and inflammatory response induced by hemodynamic changes preceding intracranial aneurysm formation: experimental study in rats.

OBJECT: Intracranial aneurysms are the leading cause of subarachnoid hemorrhage, which is associated with high morbidity and mortality rates. Despite advances in the microsurgical and endovascular treatment of intracranial aneurysms, little is known about the mechanisms by which they originate, grow, and rupture. To clarify the series of early events leading to formation of intracranial aneurysms, the authors compared aneurysmal morphological changes on vascular corrosion casts with parallel pathological changes in the cerebral arteries of rats. METHODS: The authors induced cerebral aneurysms by renal hypertension and right common carotid artery ligation in 40 male Sprague-Dawley rats; 10 intact rats served as the controls. The anterior cerebral artery-olfactory artery bifurcation was assessed morphologically by using vascular corrosion casts of Batson plastic reagent and immunohistochemically by using antibodies against endothelial nitric oxide synthase, alpha-smooth muscle actin, macrophages, and matrix metalloproteinase-9. RESULTS: Surgically treated rats manifested different degrees of aneurysmal changes. Based on these staged changes, the authors propose that the formation of intracranial aneurysms starts with endothelial injury at the apical intimal pad (Stage I); this leads to the formation of an inflammatory zone (Stage II), followed by a partial tear or defect in the inflammatory zone. Expansion of this defect forms the nidus of the intracranial aneurysm (Stage III). CONCLUSIONS: This is the first study to demonstrate the in vivo mechanisms of intracranial aneurysm formation. The inflammatory response that follows endothelial injury is the basic step in the pathogenesis of these lesions. In this study the investigators have expanded the understanding of the origin of intracranial aneurysms and have contributed to the further development of measures to prevent and treat aneurysms.