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Adipocyte-rich omentum offers "good soil" for disseminating ovarian cancer (OvCa), contributing to therapeutic difficulty. However, little is understood about the association between adipocytes and tumor growth at peritoneal dissemination site. Herein, we report the induction of adipocyte dedifferentiation by OvCa cells and pro-tumorigenic effects of resulted adipocyte-derived fibroblasts. We confirmed that malignant ascites promoted the dedifferentiation of the primary human adipocytes obtained from surgical omental specimen into omental adipocyte-derived fibroblast (O-ADF) that possess both mesenchymal stem cell and myofibroblast-like features. This promotion of dedifferentiation by malignant ascites was blocked by addition of Wnt signaling inhibitor. The effects of dedifferentiated adipocytes in proliferation and migration of OvCa cells were analyzed with in vitro co-culturing experimental models and in vivo mice model, and we demonstrated that OvCa cell lines showed enhanced proliferative characteristics, as well as increased migratory abilities upon co-culturing with O-ADF. Additionally, exogenous transforming growth factor-β1 augmented desmoplastic morphological change of O-ADF, leading to higher proliferative ability. Our results suggest that OvCa cells promote dedifferentiation of peritoneal adipocytes by activating Wnt/β-catenin signaling, and generated O-ADFs exhibit pro-tumoral hallmarks. This article is protected by copyright. All rights reserved.

In normal development, the rate of cell differentiation is tightly controlled and critical for normal development and stem cell differentiation. However, the underlying mechanisms regulating the rate of the differentiation are unknown, and manipulation of the rate of the stem cell differentiation is currently difficult. Here we show that activation of protein kinase A (PKA) accelerates the rate of mouse embryonic stem cell (ESC) differentiation through an early loss of ESC pluripotency markers and early appearance of mesodermal and other germ layer cells. The activation of PKA hastened differentiation by increasing the expression of a histone H3 lysine 9 (H3K9) dimethyltransferase, G9a protein, and the level of a negative epigenetic histone mark, H3K9 dimethylation (H3K9me2), in the promoter regions of the pluripotency markers Nanog and Oct4. These results elucidate a novel role of PKA on ESC differentiation and offer an experimental model for controlling the rate of ESC differentiation.

Epithelial ovarian cancer (EOC) is considered to secrete various factors in order to promote peritoneal dissemination through cell-to-cell interaction between cancer and mesothelial cells. We previously revealed that TGF-β secreted from EOC induces normal human peritoneal mesothelial cells (HPMCs) to differentiate into cancer-associated mesothelial cells (CAMCs). However, the relationship between tumor cells and CAMCs in EOC is still unclear. We hypothesized that CAMCs also secrete chemokines that attract cancer cells and induce peritoneal dissemination of EOC. We examined chemokines secreted from HPMCs and CAMCs by human chemokine array, and revealed that conditioned medium of CAMCs (CAMCs-CM) included many types of chemokines. The signals of CCL2 were the highest compared with other chemokines. The secretion and relative expression of CCL2 were significantly higher in CAMCs. Recombinant CCL2 promoted trans-mesothelial migration of HPMCs and the migration and invasion by EOC cells. In addition, CCL2 secreted from CAMCs promoted invasion of EOC cells. Furthermore, the neutralizing antibody of CCL2 reduced invasion by EOC. Clinical outcomes of patients whose tissue expressed higher CCR2 were significantly poorer than in patients whose tissue expression was lower. CCL2 activated the phosphorylation of p38 mitogen-activated protein kinase (MAPK). In addition, CAMCs-CM activated the p38 MAPK pathway. Phosphorylation of p38 MAPK reduced with the presence of neutralizing antibody of CCL2. In conclusion, these data indicate CCL2 in CAMCs-CM promoted the malignant potential of EOC. CCL2 plays a crucial role in the tumor microenvironment of EOC.

The mesothelium, covered by a continuous monolayer of mesothelial cells, is the first protective barrier against metastatic ovarian cancer. However, mesothelial cells release tumor-promoting factors that accelerate the process of peritoneal metastasis. We identified cancer-associated mesothelial cells (CAMs) that had tumor-promoting potential. Here, we found that plasminogen activator inhibitor-1 (PAI-1) induced the formation of CAMs, after which CAMs increasingly secreted the oncogenic factors interleukin-8 (IL-8) and C-X-C motif chemokine ligand 5 (CXCL5), further promoting the metastasis of ovarian cancer cells in a feedback loop. After the formation of CAMs, PAI-1 activated the nuclear factor kappa B (NFκB) pathway in the CAMs, thus transcriptionally upregulating the expression of the downstream NFκB targets IL-8 and CXCL5. Moreover, PAI-1 correlated with peritoneal metastasis in ovarian cancer patients and indicated a poor prognosis. In both ex vivo and in vivo models, after PAI-1 expression was knocked down, the metastasis of ovarian cancer cells decreased significantly. Therefore, targeting PAI-1 may provide a potential target for future therapeutics to prevent the formation of CAMs and alleviate peritoneal metastasis in ovarian cancer patients., ファイル公開：2020-02-01

Electricity plays an important role in modern societies, with lighting and illumination accounting for approximately one-fifth of the global demand for electricity. Haworthia obtusa has the remarkable ability to collect solar light through a so-called ‘window’ which allows it to photosynthesise in the dark. Inspired by this unique characteristic, we developed a novel lighting system that does not use electricity. The ‘window’ of H. obtusa is replicated using a scattering medium that collects solar light and guides it to an optical fibre. The optical fibre then carries the light indoors, where illumination is needed. The efficacy of this unique lighting system was confirmed both numerically and experimentally. The developed system should help in lowering energy consumption.

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.

Plasma-activated medium (PAM) is a novel chemotherapy that induces reactive oxygen species (ROS) and cell death in a wide range of cancer cell types, suggesting that PAM may be a promising therapeutic option for cancer treatment. However, dose response experiments suggest that PAM sensitivity is cell line specific. We examined the sensitivities of three glioblastoma cell lines to PAM, and found a wide variation in cell killing that was linked to differences in PAM induced ROS and apoptosis. These results indicate that the PAM sensitivity of glioblastoma cells, and potentially cancer cells more generally, is heterogeneous and likely to be dependent on the regulation of apoptosis and antioxidant pathways in target cells.

Viral gene delivery is one of the most versatile techniques for elucidating the mechanisms underlying brain dysfunction, such as neuropsychiatric disorders. Due to the complexity of the brain, expression of genetic tools, such as channelrhodopsin and calcium sensors, often has to be restricted to a specified cell type within a circuit implicated in these disorders. Only a handful of promoters targeting neuronal subtypes are currently used for viral gene delivery. Here, we isolated conserved promoter regions of several subtype-specific genes from the macaque genome and investigated their functionality in the mouse brain when used within lentiviral vectors (LVVs). Immunohistochemical analysis revealed that transgene expression induced by the promoter sequences for somatostatin (SST), cholecystokinin (CCK), parvalbumin (PV), serotonin transporter (SERT), vesicular acetylcholine transporter (vAChT), substance P (SP) and proenkephalin (PENK) was largely colocalized with specific markers for the targeted neuronal populations. Moreover, by combining these results with in silico predictions of transcription factor binding to the isolated sequences, we identified transcription factors possibly underlying cell-type specificity. These findings lay a foundation for the expansion of the current toolbox of promoters suitable for elucidating these neuronal phenotypes.

Non-thermal atmospheric pressure plasma has been widely studied in recent years in many fields, including cancer treatment. However, its efficiency for inducing apoptosis sometimes varies depending on the cell species and experimental conditions. The aim of this study was to elucidate what causes these differences in responses to plasma treatment. Using four ovarian cancer cell lines, the cell density had a markedly negative impact on the proliferation inhibition rate (PIR) and it was more obvious in OVCAR-3 and NOS2 cells. Furthermore, TOV21G and ES-2 cells were drastically sensitive to plasma‑activated medium (PAM) compared with the other two cell lines. We demonstrated that the proportion of reactive oxygen species and cell number had a marked impact on the effect of PAM against ovarian cancer cells. Additionally it was suggested that the morphological features of cells were also closely related to the sensitivity of cancer cells to the plasma treatment.

There is an intensive need for the development of novel drugs for the treatment of epithelial ovarian cancer (EOC), the most lethal gynecologic malignancy due to the high recurrence rate. TP53 mutation is a common event in EOC, particularly in high-grade serous ovarian cancer, where it occurs in more than 90% of cases. Recently, PRIMA-1 and PRIMA‑1MET (p53 reactivation and induction of massive apoptosis and its methylated form) were shown to have an antitumor effect on several types of cancer. Despite that PRIMA-1MET is the first compound evaluated in clinical trials, the antitumor effects of PRIMA-1MET on EOC remain unclear. In this study, we investigated the therapeutic potential of PRIMA-1MET for the treatment of EOC cells. PRIMA-1MET treatment of EOC cell lines (n=13) resulted in rapid apoptosis at various concentrations (24 h IC50 2.6-20.1 µM). The apoptotic response was independent of the p53 status and chemo-sensitivity. PRIMA‑1MET treatment increased intracellular reactive oxygen species (ROS), and PRIMA-1MET-induced apoptosis was rescued by an ROS scavenger. Furthermore, RNA expression analysis revealed that the mechanism of action of PRIMA‑1MET may be due to inhibition of antioxidant enzymes, such as Prx3 and GPx-1. In conclusion, our results suggest that PRIMA-1MET represents a novel therapeutic strategy for the treatment of ovarian cancer irrespective of p53 status and chemo-sensitivity.

The lateral hypothalamus (LH), which plays a role in homeostatic functions such as appetite regulation, is also linked to arousal and motivational behavior. However, little is known about how these components are encoded in the LH. Thus cynomolgus monkeys were conditioned with two distinct contexts, i.e., an appetitive context with available rewards and an aversive context with predicted air puffs. Different LH neuron groups encoded different degrees of expectation, predictability, and risks of rewards in a specific manner. A nearly equal number of one-third of the recorded LH neurons showed a positive or negative correlation between their response to visual conditioned stimuli (CS) that predicted the probabilistic delivery of rewards (0%, 50%, and 100%) and the associative values. For another one-third of recorded neurons, a nearly equal number showed a positive or negative correlation between their responses to rewards [appetitive unconditioned stimulus (US)] and reward predictability. Some neurons exhibited their highest or lowest trace-period responses in the 50% reward trials. These response modulations were represented independently and overlaid on a consistent excitatory or inhibitory response across the conditioning events. LH neurons also showed consistent responses in the aversive context. However, the responses to aversive conditioning events depending on the air puff value and predictability were less common. The multifaceted modulation of consistent activity related to outcome predictions may reflect motivational and arousal signals. Furthermore, it may underlie the role the LH plays in the integration and relay of signals to cortices for adaptive and goal-directed physiological and behavioral responses to environmental changes. NEW & NOTEWORTHY The lateral hypothalamus (LH) is implicated in motivational and arousal behavior; however, the detailed information carried by single LH neurons remains unclear. We demonstrate that primate LH neurons encode multiple combinations of signals concerning different degrees of expectation, appreciation, and uncertainty of rewards in consistent responses across conditioning events and between different contexts. This multifaceted modulation of activity may underlie the role of the LH as a critical node integrating motivational signals with arousal signals.

Treatment of peritoneal carcinomatosis arising from gastric cancer remains a considerable challenge. In recent years, the anticancer effect of nonequilibrium atmospheric pressure plasma (NEAPP) has been reported in several cancer cell lines. Use of NEAPP may develop into a new class of anticancer therapy that augments surgery, chemotherapy, and radiotherapy. Gastric cancer cells were assessed for changes in cell morphology and rate of proliferation after treatment with NEAPP-exposed medium (PAM). To explore the functional mechanism, caspase 3/7, annexin V, and uptake of reactive oxygen species (ROS) were evaluated, along with the effect of the ROS scavenger N-acetylcysteine (NAC). PAM treatment for 24 h affected cell morphology, suggestive of induction of apoptosis. PAM cytotoxicity was influenced by the time of exposure to PAM, the type of cell line, and the number of cells seeded. Cells treated with PAM for 2 h demonstrated activated caspase 3/7 and an increased proportion of annexin V-positive cells compared with untreated cells. Additionally, ROS uptake was observed in PAM-treated cells, whereas NAC reduced the cytotoxicity induced by PAM presumably through reduction of ROS uptake. Furthermore, CD44 variant 9, which reportedly leads to glutathione synthesis and suppresses stress signaling of ROS, was overexpressed in PAM-resistant cells. PAM treatment induced apoptosis of gastric cancer cells through generation and uptake of ROS. Local administration of PAM could develop into an option to treat peritoneal carcinomatosis.

Aim: Existing evidence suggests that endothelial lipase (EL) plays an important role in high-densitylipoprotein (HDL) metabolism. Because rabbits are a useful animal model for the study of human lipid metabolism and atherosclerosis, we characterized rabbit EL (rEL) expression and investigated its relationship with plasma HDL levels in normal and hyperlipidemic rabbits.Methods: We cloned the rEL cDNA and analyzed the EL tissue expression using Northern blotting, real-time RT-PCR, Western blotting, and in situ hybridization. We evaluated the effects of rEL antisense on plasma HDL levels.Results: We found that rEL mRNA was highly expressed in cholesterol synthesis-related organs, including the liver, testis, and adrenal along with its expression in the lung, kidney, bone marrow, and small intestine. Interestingly, Watanabe heritable hyperlipidemic (WHHL) rabbits, a model of human familial hypercholesterolemia, had lower plasma levels of HDLs than normal rabbits. The plasma HDL levels in WHHL rabbits were inversely associated with high levels of plasma rEL proteins and hepatic expression of rEL mRNA. Injection of rEL-specific antisense oligonucleotides into rabbits resulted in the elevation of plasma large HDLs. Furthermore, we demonstrated that rEL mRNA was expressed by both endothelial cells and macrophages in the lesions of aortic atherosclerosis of WHHL rabbits.Conclusions: rEL is expressed in multiple tissues and may have many physiological and pathophysiological functions, such as in the regulation of cholesterol metabolism and atherosclerosis. Our results suggest that EL is an important regulator of plasma HDL levels in rabbits.

The authors performed a pathological examination of a 5-year-old female laboratory Japanese monkey who developed cortical blindness and epileptic seizures. Generalized, tonic-clonic seizures started to occur during behavioral training to get the animal to enter a carrying cage for future psychological experiments. Blindness was suspected because of a lack of approaching behavior toward foods such as fruits. Although the monkey was extensively treated with anticonvulsants, the clinical signs did not improve. An increased serum creatine phosphokinase (CPK) level and bilateral occipital brain atrophy were detected. Histopathologically, a severe degree of cerebromalacia was detected bilaterally in the occipital lobe, and necrosis and gliosis were seen mainly in the temporal lobe. Focal inflammation was found in the meninges. No other changes were observed in other nervous tissues or organs, and no signs of a parasitic or viral infection were found in the systemic organs. Spontaneously occurring lesions in the central nervous system have been rarely reported in laboratory monkeys. In the present case, the cause of cerebromalacia could not be confirmed, but the relationship between symptoms such as abnormal vision and the presence of brain lesions was distinct. The authors believe that this case is a valuable historical control case for the laboratory Japanese macaque.

Although it has been demonstrated that statins stabilize atherosclerotic lesions in animal models of advanced atherosclerosis, there is little evidence to suggest that statins have a preventive effect on plaque rupture itself. In the present study, we examined the effect of fluvastatin on plaque disruption using a simple and quick method of plaque disruption in carotid artery lesions in apolipoprotein E-deficient mice. Male apolipoprotein E-deficient mice received normal chow and underwent ligation of the left common carotid artery just proximal to its bifurcation. Four weeks later, a polyethylene cuff was placed around the artery immediately proximal to the ligation site. Fluvastatin (10 mg/kg per day) was given by oral gavage every day starting at 3 days before cuff placement. The administration of fluvastatin suppressed atherosclerotic plaque disruption accompanied by luminal thrombi by 31.5% compared with controls at 4 days after the cuff was placed at the ligated carotid artery. Fluvastatin administration decreased matrix metalloproteinase-9 expression, gelatinolytic activity, endothelial adhesion molecules expression and neutrophil infiltration, and increased type I collagen content in the cuffed region. In summary, fluvastatin was found to prevent plaque disruption through pleiotropic effect on acute inflammation in an animal model using apolipoprotein E-deficient mice.

Cathepsins are cysteine proteases that participate in various types of tissue remodeling. However, their expressions during myocardial remodeling have not been examined. In this study, we investigated their expressions in the left ventricular (LV) myocardium of rats and humans with hypertension-induced LV hypertrophy or heart failure (HF). Real-time PCR and immunoblot analysis revealed that the abundance of cathepsin S mRNA or protein in the LV tissues was greater in rats or humans with HF than in those with hypertrophy or in control subjects. Immunostaining showed that cathepsin S was localized predominantly to cardiac myocytes and coronary vascular smooth muscle cells, but also overlapped in part with macrophages. Elastic lamina fragmentations significantly increased in the LV intramyocardial coronary arteries of HF rats. The amount of elastolytic activity in the extract of the LV myocardium was markedly increased for HF rats compared with controls, and this activity was mostly because of cathepsin S. Although the amount of elastin mRNA was increased in the LV myocardium of HF rats, the area of interstitial elastin was not. The expression of interleukin 1β was increased in the LV myocardium of HF rats, and this cytokine was found to increase the expression and activity of cathepsin S in cultured neonatal cardiomyocytes. These results suggest that cathepsin S participates in pathological LV remodeling associated with hypertension-induced HF. This protease is, thus, a potential target for therapeutics aimed at preventing or reversing cardiac remodeling.

Objective— The development of a murine model of atherosclerotic plaque rupture. Methods and Results— The left common carotid arteries of male apolipoprotein E (apoE)–deficient mice (9 weeks old) were ligated just proximal to their bifurcations. After 4 weeks on a standard diet, the mice received polyethylene cuff placement just proximal to the ligated site, and the animals were then processed for morphological studies at specific time points. Ligation of the carotid artery in apoE-deficient mice for 4 weeks induced marked intimal hyperplasia, which is a lipid- and collagen-rich lesion that contains a number of macrophages, T lymphocytes, and smooth muscle cells. Subsequently, the cuff placement evoked intraplaque hemorrhage and plaque rupture with fibrin(ogen)-positive luminal thrombus in this region accompanying a decrease in collagen content as well as an increase in apoptotic cells in the intima within a few days after cuff placement. Conclusions— We demonstrated the murine model of human plaque rupture, which is simple, fast, and highly efficient. This model would help us not only to understand the mechanism of human plaque rupture but also to assess various already-known and as-yet-unknown agents in the future.

A novel model to induce occlusive thrombus formation was developed in mice in vivo. Mice were simultaneously treated with ligation and cuff placement at the left carotid artery. At 7 days after the treatment, occlusive thrombus was observed at the intracuff region, but not in the distal and proximal regions of the cuff, and not induced by a single treatment of ligation or cuff placement. The plasma levels of von Willebrand factor (vWF), which represent the endothelial status, were significantly increased in combined treatment of ligation and cuff placement 1 day after the operation. Whereas no significant changes in plasma vWF were observed in either single treatment of ligation or cuff placement. The expression of vWF, considered to be the endothelial marker, was detected on the luminal surface distal and proximal to the cuff and the carotid artery in the single treatment groups treated with either ligation or cuff placement, but was not detected in the intracuff region. Furthermore, the binding of Griffolia Simplicifolia Lectin-I (GSL-I) and endothelial nitric oxide synthase (eNOS) expression indicating the endothelial integrity was not detected in the intracuff region. Intermittent injections of ancrod, which decreases the plasma fibrinogen, inhibited occlusive thrombus formation in the intracuff region. The expression of eNOS was detected at the distal and proximal but not the intracuff region of the carotid artery treated with ancrod. Daily administration of aspirin significantly suppressed the thrombus formation in this model. These results indicate that occlusive thrombus formation accompanied by endothelial damage or dysfunction is induced by the combined application of ligation and cuff placement at the carotid artery, and suggest that this endothelial damage or dysfunction may be one pathogenesis of thrombogenesis in this model.

Four isomers of acetoxycineoles, (trans and cis)-2- and 3-acetoxy-1,8-cineoles, were identified as the odorous components of the rhizomes of greater galangal. Their structures were confirmed by comparing the retention indices by GC and the mass spectra with those of synthesized compounds. The concentration of trans-3-acetoxy-1,8-cineole was the highest among the isomers. The isomers presented individual odor features: the (trans and cis)-2 isomers respectively exhibited woody and sweet aromas, while the (trans and cis)-3 isomers respectively showed sweet floral and camphoraceous aromas. Of these, trans-2-acetoxy-1,8-cineole seemed to have the strongest qualitative effect on the characteristic flavor of greater galangal. Keywords: Alpinia galanga Willd.; greater galangal; rhizome; flavor component; trans-2-acetoxy-1,8-cineole; cis-2-acetoxy-1,8-cineole; trans-3-acetoxy-1,8-cineole; cis-3-acetoxy-1,8-cineole

Objective Matrix metalloproteinases (MMPs) have been implicated in the process of vascular calcification. However, the exact roles of individual MMPs in vascular calcification are poorly understood. To study the putative role of MMP-2 in atherogenic calcification in vivo and in vitro , we investigate whether or not MMP-2 deficiency affects aortic atherosclerotic calcification in apolipoprotein E-deficient (Apoe −/− ) mice and cultured smooth muscle cell (SMC) calcification. Methods and results The area of calcified lesions in aortic intima was significantly larger in MMP-2 +/+ Apoe −/− mice at 45 and 60 weeks of age than in MMP-2 −/− Apoe −/− mice. In these aortic calcified atherosclerotic lesions, the expression of type II collagen, osteoprotegerin, bone morphogenetic protein (BMP)-2 and osteocalcin which are chondrocyte maker and bone-related proteins, was observed. MMP-2 deficiency reduced BMP-2 and osteocalcin expression in aortae in Apoe −/− mice at 30 weeks and 45–60-weeks-old, respectively. The expressions of MMP-2 and that of α-SMC actin were observed in chondrocyte-like cells of calcified lesions. Beta-glycerophosphate administration induced calcium deposition in MMP-2 +/+ aorta-derived cultured SMCs, and this calcium deposition was significantly suppressed in MMP-2 −/− aorta-derived cultured SMCs. Conclusions These results suggest that MMP-2 may contribute to the mechanisms of calcification associated with atherosclerosis.

Nitroalkene derivatives of nitro-linoleic acid (LNO 2 ) and nitro-oleic acid (OA-NO 2 ) are nitrated unsaturated fatty acids that can be detected in healthy human plasma, red blood cells and urine. It has been shown that nitroalkenes have potent anti-inflammatory properties in multiple disease models. In the present study, we are the first to investigate the apoptotic effects of nitroalkenes in rat aortic smooth muscle cells (RASMCs). We observed that nitroalkenes induce RASMCs apoptosis in a dose-dependent manner. In addition, nitroalkenes stimulate extrinsic caspase-8 and intrinsic caspase-9 activity to trigger the caspase-3 apoptotic signaling cascade, resulting in RASMCs death. Furthermore, the pro-apoptotic protein, Bad was upregulated and antiapoptotic protein, Bcl-xl was downregulated during nitroalkene-induced apoptosis. These results demonstrate that nitroalkenes can induce RASMCs apoptosis via stimulation of caspase activity and the regulation of apoptotic protein expression levels.

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.

Matrix metalloproteinases (MMPs) have been implicated in the process of neovascularization. However, the exact roles of individual MMPs in vessel formation are poorly understood. To study the putative role of MMP-2 in ischemia-induced neovascularization, a hindlimb ischemia model was applied to MMP-2 +/+ and MMP-2 −/− mice. Serial laser Doppler blood-flow analysis revealed that the recovery of the ischemic/normal blood-flow ratio in MMP-2 −/− young and old mice remained impaired throughout the follow-up period. At day 35, microangiography and anti– l -lectin immunohistochemical staining revealed lesser developed collateral vessels and capillary formation in both old and young MMP-2 −/− mice compared with the age-matched MMP-2 +/+ mice. An aortic-ring culture assay showed a markedly impaired angiogenic response in MMP-2 −/− mice, which was partially recovered by supplementation of the culture medium with recombinant MMP-2. Aorta-derived endothelial cells or bone marrow–derived endothelial progenitor cell (EPC)-like c-Kit + cells from MMP-2 −/− showed marked impairment of invasive or/and proliferative abilities. At day 7, plasma and ischemic tissues of vascular endothelial growth factor protein were reduced in MMP-2 −/− . Flow cytometry showed that the numbers of EPC-like CD31 + c-Kit + cells in peripheral blood markedly decreased in MMP-2–deficient mice. Transplantation of bone marrow–derived mononuclear cells from MMP-2 +/+ mice restored neovascularization in MMP-2 −/− young mice. These data suggest that MMP-2 deficiency impairs ischemia-induced neovascularization through a reduction of endothelial cell and EPC invasive and/or proliferative activities and EPC mobilization.

Objective— Although it has been reported that matrix metalloproteinase (MMP)-2 is a major proteinase in atherosclerotic plaque lesions, there is no direct evidence of the role of MMP-2 in atherosclerotic lesion formation. In the present study we determined the role of MMP-2 in atherosclerosis plaque development using apolipoprotein E-deficient (apoE −/− ) mice. Methods and Results— To generate MMP-2–deficient, apoE-deficient mice (MMP-2 −/− :apoE −/− ), MMP-2 −/− mice were crossed with apoE −/− mice. After 8 weeks of feeding with a lipid-rich diet, morphological and biochemical studies of the aortic sinus and arch were conducted. A significant reduction of the atherosclerotic plaque in the aortic sinus and arch with the decrease in smooth muscle cell-positive area was observed in MMP-2 −/− :apoE −/− mice compared with that of MMP-2 +/+ :apoE −/− mice. Macrophage- and collagen-positive areas were less in aortic sinus but not in aortic arch in MMP-2 −/− :apoE −/− mice. There was no difference of MMP-9 mRNA expression in the plaque lesion between the 2 genotypes. A much lower level of mRNA expression of TIMP-1 and TIMP-2 was detected in the atherosclerotic plaque lesions of MMP-2 −/− :apoE −/− mice than in those of MMP-2 +/+ :apoE −/− mice. Conclusions— MMP-2 contributes to the development of atherosclerosis in apoE −/− mice.

Smooth muscle cell (SMC) migration from the tunica media to the intima, a key event in neointimal formation, requires proteolytic degradation of elastin-rich extracellular matrix barriers. Although cathepsin S (Cat S) is overexpressed in atherosclerotic and neointimal lesions, its exact role in SMC behavior remains primarily unresolved. We examined the involvement of Cat S on SMC migration through an extracellular matrix barrier and its localization in SMCs. A selective Cat S inhibitor and the endogenous inhibitor cystatin C significantly attenuated SMC invasion across elastin gel. Western blotting and cell surface biotinylation analysis demonstrated localization of the 28-kd active form of Cat S on the SMC surface, consistent with its role in the proteolysis of subcellular matrices. Treatment with interferon-gamma or interleukin-beta1 significantly augmented the ability of SMC membranes to degrade elastin along with a significant increase in the level of active Cat S compared with controls. Immunofluorescence and confocal microscopy showed a punctuated pattern of Cat S clusters at the periphery of SMCs; further studies demonstrated partial co-localization of Cat S and integrin alphanubeta3 at the cell surfaces. These findings demonstrate that active Cat S co-localizes with integrin alphanubeta3 as a receptor on the SMC surface, playing an important role in the invasive behavior of SMCs.

Objective— Although we recently showed that the administration of catechins reduced the neointimal formation in a rat balloon-injury model, the precise molecular mechanisms are largely unknown. In the present study, we tried to determine these mechanisms using an in vitro SMC invasion system. Methods and Results— Boyden chamber assay was used to examine the effect of catechins on the invasive behavior of SMCs. The invasive activity of SMCs through collagen gel was restrained by EGCG in a concentration-dependent manner. The data from gelatin and collagen zymography and Western blot revealed that EGCG blocks the activation of pro-matrix metalloproteinase (MMP)-2 during an invasion assay and in the conditioned medium of cultured SMCs as well as the activities of MMP-2 and membrane type 1-MMP (MT1-MMP) even at 0.1 to 0.3 μmol/L of EGCG. EGCG was found to restrain MT1-MMP cat –dependent pro–MMP-2 activation. EGCG upregulated the expression of tissue inhibitor of MMP-2 (TIMP-2) protein. Reverse zymography showed that the increased TIMP-2 to expression was validated by an increased activity. The data from decreased TIMP-2 activity using its siRNA suggested that upregulation of TIMP-2 expression may be one of the major mechanisms for inhibition of SMC invasion by EGCG. Conclusions— These results indicate that EGCG targets multiple MMP-mediated SMC cellular events and provides a new major mechanism for the SMC invasion through upregulation of TIMP-2 expression to modulate MMP activity.

It has been suggested on the basis of previous studies involving functional MRI (fMRI) and single-neuron recording that neurons of the supplementary eye field (SEF) and anterior cingulate cortex (ACC) monitor conflict. To test this idea, we carried out microelectrode recording in monkeys performing a color-conditional eye movement task in which red and green cues instructed leftward and rightward saccades, respectively. In a variant inducing conflict by spatial incompatibility, the cue was presented either at the location of the target (no conflict) or opposite the location of the target (conflict). In a variant inducing conflict by reversal, the foveal cue either remained one color (no conflict) or reversed color after 100 ms (conflict), with the monkey required to follow the instruction conveyed by the second color. In both tasks, conflict was evident in behavioral measures (reduced percent correct and slowed reaction time) and in physiological measures (reduced strength of directional activity among direction-selective neurons). In the SEF, there was a tendency for neurons to fire more strongly on trials involving conflict, but this effect took the form of modulation of task-related activity among direction-selective neurons, not of a pure conflict-monitoring signal. In the ACC, there was no conflict-related enhancement. These results are incompatible with the idea that the SEF and ACC contain populations of neurons specialized for monitoring conflict.

Neurons in the lateral intraparietal area, frontal eye field, and superior colliculus exhibit a pattern of activity known as remapping. When a salient visual stimulus is presented shortly before a saccade, the representation of that stimulus is updated, or remapped, at the time of the eye movement. This updating is presumably based on a corollary discharge of the eye movement command. To investigate whether visual areas also exhibit remapping, we recorded from single neurons in extrastriate and striate cortex while monkeys performed a saccade task. Around the time of the saccade, a visual stimulus was flashed either at the location occupied by the neuron's receptive field (RF) before the saccade (old RF) or at the location occupied by it after the saccade (new RF). More than half (52%) of V3A neurons responded to a stimulus flashed in the new RF even though the stimulus had already disappeared before the saccade. These neurons responded to a trace of the flashed stimulus brought into the RF by the saccade. In 16% of V3A neurons, remapped activity began even before saccade onset. Remapping also was observed at earlier stages of the visual hierarchy, including in areas V3 and V2. At these earlier stages, the proportion of neurons that exhibited remapping decreased, and the latency of remapped activity increased relative to saccade onset. Remapping was very rare in striate cortex. These results indicate that extrastriate visual areas are involved in the process of remapping.