TRIM25-mediated XRCC1 ubiquitination accelerates atherosclerosis by inducing macrophage M1 polarization and programmed death.

BACKGROUND: Macrophage-mediated cleaning up of dead cells is a crucial determinant in reducing coronary artery inflammation and maintaining vascular homeostasis. However, this process also leads to programmed death of macrophages. So far, the role of macrophage death in the progression of atherosclerosis remains controversial. Also, the underlying mechanism by which transcriptional regulation and reprogramming triggered by macrophage death pathways lead to changes in vascular inflammation and remodeling are still largely unknown. TRIM25-mediated RIG-I signaling plays a key role in regulation of macrophages fate, however the role of TRIM25 in macrophage death-mediated atherosclerotic progression remains unclear. This study aims to investigate the relationship between TRIM25 and macrophage death in atherosclerosis. METHODS: A total of 34 blood samples of patients with coronary stent implantation, including chronic total occlusion (CTO) leisions (n = 14) or with more than 50% stenosis of a coronary artery but without CTO leisions (n = 20), were collected, and the serum level of TRIM25 was detected by ELISA. Apoe-/- mice with or without TRIM25 gene deletion were fed with the high-fat diet (HFD) for 12 weeks and the plaque areas, necrotic core size, aortic fibrosis and inflammation were investigated. TRIM25 wild-type and deficient macrophages were isolated, cultured and stimulated with ox-LDL, RNA-seq, real-time PCR, western blot and FACS experiments were used to screen and validate signaling pathways caused by TRIM25 deletion. RESULTS: Downregulation of TRIM25 was observed in circulating blood of CTO patients and also in HFD-induced mouse aortas. After HFD for 12 weeks, TRIM25-/-ApoeE-/- mice developed smaller atherosclerotic plaques, less inflammation, lower collagen content and aortic fibrosis compared with TRIM25+/+ApoeE-/- mice. By RNA-seq and KEGG enrichment analysis, we revealed that deletion of TRIM25 mainly affected pyroptosis and necroptosis pathways in ox-LDL-induced macrophages, and the expressions of PARP1 and RIPK3, were significantly decreased in TRIM25 deficient macrophages. Overexpression of TRIM25 promoted M1 polarization and necroptosis of macrophages, while inhibition of PARP1 reversed this process. Further, we observed that XRCC1, a repairer of DNA damage, was significantly upregulated in TRIM25 deficient macrophages, inhibiting PARP1 activity and PARP1-mediated pro-inflammatory change, M1 polarization and necroptosis of macrophages. By contrast, TRIM25 overexpression mediated ubiquitination of XRCC1, and the inhibition of XRCC1 released PARP1, and activated macrophage M1 polarization and necroptosis, which accelerated aortic inflammation and atherosclerotic plaque progression. CONCLUSIONS: Our study has uncovered a crucial role of the TRIM25-XRCC1Ub-PARP1-RIPK3 axis in regulating macrophage death during atherosclerosis, and we highlight the potential therapeutic significance of macrophage reprogramming regulation in preventing the development of atherosclerosis.

(-)-Epigallocatechin gallate alleviates chronic unpredictable mild stress-induced depressive symptoms in mice by regulating the mTOR autophagy pathway and inhibiting NLRP3 inflammasome activation.

Depression is a global public health issue that is widely studied due to the large number of people it affects and its serious consequences. Clinical studies have shown that regular tea consumption may reduce depression risk. (-)-Epigallocatechin gallate (EGCG), the main tea polyphenol, was observed to alleviate depression, but the underlying mechanism has not been elucidated. In this study, chronic unpredictable mild stress (CUMS) was used to induce depression-like behavior in mice, and behavioral tests, such as sucrose preference test and forced swim test, were performed. Then, ELISA, western blot and QT-PCR tests were used to assess the expression of the key components of the NLRP3 inflammasome and its downstream inflammatory effectors (e.g., IL-1ß, IL-18), autophagy markers (Beclin-1, LC3, P62) and apoptosis markers (Bax, Bcl-2) in mouse brain tissues. Changes in serum lipid levels were also assessed. EGCG alleviated CUMS-induced depression-like behavioral changes in mice, reduced activation of the NLRP3 inflammasome, inhibited the mTOR signaling pathway, restored autophagy levels, reduced apoptosis marker expression and attenuated abnormal changes in blood lipid levels. Our study demonstrates that EGCG exerts antidepressive effects through multiple mechanisms, providing new insight into the pathological mechanism of depression and laying the foundation for the development of new therapeutic measures.

Epigenetically altered macrophages promote development of diabetes-associated atherosclerosis.

Background: Atherosclerosis (AS) risk is elevated in diabetic patients, but the underlying mechanism such as involvement of epigenetic control of foam macrophages remains unclear. We have previously shown the importance of immune regulation on endothelial cells to AS development in diabetes. In this study, we examined the hypothesis that diabetes may promote AS through modification of the epigenetic status of macrophages. Methods: We employed the Laser Capture Microdissection (LCM) method to evaluate the expression levels of key epigenetic regulators in both endothelial cells and macrophages at the AS lesions of patients. We then assessed the correlation between the significantly altered epigenetic regulator and serum levels of low-density Lipoprotein (LDL), triglycerides (TRIG) and high-density Lipoprotein (HDL) in patients. In vitro, the effects of high glucose on glucose utilization, lactate production, succinate levels, oxygen consumption and polarization in either undifferentiated or differentiated bone marrow-derived macrophages (BMDMs) were analyzed. The effects of depleting this significantly altered epigenetic regulator in macrophages on AS development were assessed in AS-prone diabetic mice. Results: Histone deacetylase 3 (HDAC3) was identified as the most significantly altered epigenetic regulator in macrophages from the AS lesions in human diabetic patients. The levels of HDAC3 positively correlated with high serum LDL and TRIG, as well as low serum HDL. High glucose significantly increased glucose utilization, lactate production, succinate levels and oxygen consumption in cultured macrophages, and induced proinflammatory M1-like polarization. Macrophage depletion of HDAC3 significantly attenuated AS severity in AS-prone diabetic mice. Conclusion: Epigenetically altered macrophages promote development of diabetes-associated AS, which could be prevented through HDAC3 depletion.

Young bone marrow transplantation prevents aging-related muscle atrophy in a senescence-accelerated mouse prone 10 model.

BACKGROUND: Young bone marrow transplantation (YBMT) has been shown to stimulate vascular regeneration in pathological conditions, including ageing. Here, we investigated the benefits and mechanisms of the preventive effects of YBMT on loss of muscle mass and function in a senescence-associated mouse prone 10 (SAMP10) model, with a special focus on the role of growth differentiation factor 11 (GDF-11). METHODS: Nine-week-old male SAMP10 mice were randomly assigned to a non-YBMT group (n = 6) and a YBMT group (n = 7) that received the bone marrow of 8-week-old C57BL/6 mice. RESULTS: Compared to the non-YBMT mice, the YBMT mice showed the following significant increases (all P < 0.05 in 6-7 mice): endurance capacity (>61.3%); grip strength (>37.9%), percentage of slow myosin heavy chain fibres (>14.9-15.9%). The YBMT also increased the amounts of proteins or mRNAs for insulin receptor substrate 1, p-Akt, p-extracellular signal-regulated protein kinase1/2, p-mammalian target of rapamycin, Bcl-2, peroxisom proliferator-activated receptor-γ coactivator (PGC-1α), plus cytochrome c oxidase IV and the numbers of proliferating cells (n = 5-7, P < 0.05) and CD34+/integrin-α7+ muscle stem cells (n = 5-6, P < 0.05). The YMBT significantly decreased the levels of gp91phox, caspase-9 proteins and apoptotic cells (n = 5-7, P < 0.05) in both muscles; these beneficial changes were diminished by the blocking of GDF-11 (n = 5-6, P < 0.05). An administration of mouse recombinant GDF-11 improved the YBMT-mediated muscle benefits (n = 5-6, P < 0.05). Cell therapy with young bone marrow from green fluorescent protein (GFP) transgenic mice exhibited GFP+ myofibres in aged muscle tissues. CONCLUSIONS: These findings suggest that YBMT can prevent muscle wasting and dysfunction by mitigating apoptosis and proliferation via a modulation of GDF-11 signalling and mitochondrial dysfunction in SAMP10 mice.

Kinetic Characteristics of Curcumin and Germacrone in Rat and Human Liver Microsomes: Involvement of CYP Enzymes.

Curcumin and germacrone, natural products present in the Zingiberaceae family of plants, have several biological properties. Among these properties, the anti-NSCLC cancer action is noteworthy. In this paper, kinetics of the two compounds in rat liver microsomes (RLMs), human liver microsomes (HLMs), and cytochrome P450 (CYP) enzymes (CYP3A4, 1A2, 2E1, and 2C19) in an NADPH-generating system in vitro were evaluated by UP-HPLC-MS/MS (ultrahigh-pressure liquid chromatography-tandem mass spectrometry). The contents of four cytochrome P450 (CYP) enzymes, adjusting by the compounds were detected using Western blotting in vitro and in vivo. The t1/2 of curcumin was 22.35 min in RLMs and 173.28 min in HLMs, while 18.02 and 16.37 min were gained for germacrone. The Vmax of curcumin in RLMs was about 4-fold in HLMs, meanwhile, the Vmax of germacrone in RLMs was similar to that of HLMs. The single enzyme t1/2 of curcumin was 38.51 min in CYP3A4, 301.4 min in 1A2, 69.31 min in 2E1, 63.01 min in 2C19; besides, as to the same enzymes, t1/2 of germacrone was 36.48 min, 86.64 min, 69.31 min, and 57.76 min. The dynamic curves were obtained by reasonable experimental design and the metabolism of curcumin and germacrone were selected in RLMs/HLMs. The selectivities in the two liver microsomes differed in degradation performance. These results meant that we should pay more attention to drugs in clinical medication-drug and drug-enzyme interactions.

Deficiency of cysteinyl cathepsin K suppresses the development of experimental intimal hyperplasia in response to chronic stress.

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.

Cathepsin S Deficiency Mitigated Chronic Stress-Related Neointimal Hyperplasia in Mice.

Background Exposure to chronic psychosocial stress is a risk factor for atherosclerosis-based cardiovascular disease. We previously demonstrated the increased expressions of cathepsin S (CatS) in atherosclerotic lesions. Whether CatS participates directly in stress-related neointimal hyperplasia has been unknown. Methods and Results Male wild-type and CatS-deficient mice that underwent carotid ligation injury were subjected to chronic immobilization stress for morphological and biochemical studies at specific times. On day 14 after stress/surgery, stress enhanced the neointima formation. At the early time points, the stressed mice had increased plaque elastin disruption, cell proliferation, macrophage accumulation, mRNA and/or protein levels of vascular cell adhesion molecule-1, angiotensin II type 1 receptor, monocyte chemoattractant protein-1, gp91phox, stromal cell-derived factor-1, C-X-C chemokine receptor-4, toll-like receptor-2, toll-like receptor-4, SC 35, galectin-3, and CatS as well as targeted intracellular proliferating-related molecules (mammalian target of rapamycin, phosphorylated protein kinase B, and p-glycogen synthase kinase-3α/ß). Stress also increased the plaque matrix metalloproteinase-9 and matrix metalloproteinase-2 mRNA expressions and activities and aorta-derived smooth muscle cell migration and proliferation. The genetic or pharmacological inhibition of CatS by its specific inhibitor (Z- FL -COCHO) ameliorated the stressed arterial targeted molecular and morphological changes and stressed aorta-derived smooth muscle cell migration. Both the genetic and pharmacological interventions had no effect on increased blood pressure in stressed mice. Conclusions These results demonstrate an essential role of CatS in chronic stress-related neointimal hyperplasia in response to injury, possibly via the reduction of toll-like receptor-2/toll-like receptor-4-mediated inflammation, immune action, and smooth muscle cell proliferation, suggesting that CatS will be a novel therapeutic target for stress-related atherosclerosis-based cardiovascular disease.

Nicotine promotes atherosclerosis development in apolipoprotein E-deficient mice through α1-nAChR.

α1 Nicotinic acetylcholine receptor (α1nAChR) is an important nicotine receptor that is widely distributed in vascular smooth muscle cells, macrophages, and endothelial cells. However, the role of α1nAChR in nicotine-mediated atherosclerosis remains unclear. The administration of nicotine for 12 weeks increased the area of the atherosclerotic lesion, the number of macrophages infiltrating the plaques, and the circulating levels of inflammatory cytokines, such as interleukin-6 and tumor necrosis factor-α, in apolipoprotein E-deficient (ApoE-/- ) mice fed a high-fat diet. Nicotine also increased α1nAChR, calpain-1, matrix metalloproteinase-2 (MMP-2), and MMP-9 expression in the aortic tissue. Silencing of α1nAChR with an adenoassociated virus decreased the atherosclerotic size, lesion macrophage content, and circulating levels of inflammatory cytokines and suppressed α1nAChR, calpain-1, MMP-2, and MMP-9 expression in the nicotine group. In vitro, nicotine-induced α1nAChR, calpain-1, MMP-2, and MMP-9 expression in mouse vascular smooth muscle cells (MOVAS) and macrophages (RAW264.7), and enhanced the migration and proliferation of these cells. The silencing of α1nAChR inhibited these effects of nicotine MOVAS and RAW264.7 cells. Thus, we concluded that nicotine promoted the development of atherosclerosis partially by inducing the migration and proliferation of vascular smooth muscle cells and macrophages and inducing an inflammatory reaction. The effect of nicotine on atherogenesis may be mediated by α1nAChR-induced activation of the calpain-1/MMP-2/MMP-9 signaling pathway.

Cysteine Protease Cathepsins in Atherosclerotic Cardiovascular Diseases.

Atherosclerotic cardiovascular disease (ASCVD) is an inflammatory disease characterized by extensive arterial wall matrix protein degradation. Cysteine protease cathepsins play a pivotal role in extracellular matrix (ECM) remodeling and have been implicated in the development and progression of atherosclerosis-based cardiovascular diseases. An imbalance in expression between cathepsins (such as cathepsins S, K, L, C) and their inhibitor cystatin C may favor proteolysis of ECM in the pathogenesis of cardiovascular disease such as atherosclerosis, aneurysm formation, restenosis, and neovascularization. New insights into cathepsin functions have been made possible by the generation of knockout mice and by the application of specific inhibitors. Inflammatory cytokines regulate the expression and activities of cathepsins in cultured vascular cells and macrophages. In addition, evaluations of the possibility of cathepsins as a diagnostic tool revealed that the circulating levels of cathepsin S, K, and L, and their endogenous inhibitor cystatin C could be promising biomarkers in the diagnosis of coronary artery disease, aneurysm, adiposity, peripheral arterial disease, and coronary artery calcification. In this review, we summarize the available information regarding the mechanistic contributions of cathepsins to ASCVD.

Chronic Psychological Stress Accelerates Vascular Senescence and Impairs Ischemia-Induced Neovascularization: The Role of Dipeptidyl Peptidase-4/Glucagon-Like Peptide-1-Adiponectin Axis.

BACKGROUND: Exposure to psychosocial stress is a risk factor for cardiovascular disease, including vascular aging and regeneration. Given that dipeptidyl peptidase-4 (DPP4) regulates several intracellular signaling pathways associated with the glucagon-like peptide-1 (GLP-1) metabolism, we investigated the role of DPP4/GLP-1 axis in vascular senescence and ischemia-induced neovascularization in mice under chronic stress, with a special focus on adiponectin -mediated peroxisome proliferator activated receptor-γ/its co-activator 1α (PGC-1α) activation. METHODS AND RESULTS: Seven-week-old mice subjected to restraint stress for 4 weeks underwent ischemic surgery and were kept under immobilization stress conditions. Mice that underwent ischemic surgery alone served as controls. We demonstrated that stress impaired the recovery of the ischemic/normal blood-flow ratio throughout the follow-up period and capillary formation. On postoperative day 4, stressed mice showed the following: increased levels of plasma and ischemic muscle DPP4 and decreased levels of GLP-1 and adiponectin in plasma and phospho-AMP-activated protein kinase α (p-AMPKα), vascular endothelial growth factor, peroxisome proliferator activated receptor-γ, PGC-1α, and Sirt1 proteins and insulin receptor 1 and glucose transporter 4 genes in the ischemic tissues, vessels, and/or adipose tissues and numbers of circulating endothelial CD31+/c-Kit+ progenitor cells. Chronic stress accelerated aortic senescence and impaired aortic endothelial sprouting. DPP4 inhibition and GLP-1 receptor activation improved these changes; these benefits were abrogated by adiponectin blocking and genetic depletion. CONCLUSIONS: These results indicate that the DPP4/GLP-1-adiponectin axis is a novel therapeutic target for the treatment of vascular aging and cardiovascular disease under chronic stress conditions.

Exenatide mitigated diet-induced vascular aging and atherosclerotic plaque growth in ApoE-deficient mice under chronic stress.

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.

Dipeptidyl Peptidase-4 Regulates Hematopoietic Stem Cell Activation in Response to Chronic Stress.

BACKGROUND: DPP4 (Dipeptidyl peptidase-4)-GLP-1 (glucagon-like peptide-1) and its receptor (GLP-1R) axis has been involved in several intracellular signaling pathways. The Adrß3 (ß3-adrenergic receptor)/CXCL12 (C-X-C motif chemokine 12) signal was required for the hematopoiesis. We investigated the novel molecular requirements between DPP4-GLP-1/GLP-1 and Adrß3/CXCL12 signals in bone marrow (BM) hematopoietic stem cell (HSC) activation in response to chronic stress. METHODS AND RESULTS: Male 8-week-old mice were subjected to 4-week intermittent restrain stress and orally treated with vehicle or the DPP4 inhibitor anagliptin (30 mg/kg per day). Control mice were left undisturbed. The stress increased the blood and brain DPP4 levels, the plasma epinephrine and norepinephrine levels, and the BM niche cell Adrß3 expression, and it decreased the plasma GLP-1 levels and the brain GLP-1R and BM CXCL12 expressions. These changes were reversed by DPP4 inhibition. The stress activated BM sca-1highc-KithighCD48lowCD150high HSC proliferation, giving rise to high levels of blood leukocytes and monocytes. The stress-activated HSC proliferation was reversed by DPP4 depletion and by GLP-1R activation. Finally, the selective pharmacological blocking of Adrß3 mitigated HSC activation, accompanied by an improvement of CXCL12 gene expression in BM niche cells in response to chronic stress. CONCLUSIONS: These findings suggest that DPP4 can regulate chronic stress-induced BM HSC activation and inflammatory cell production via an Adrß3/CXCL12-dependent mechanism that is mediated by the GLP-1/GLP-1R axis, suggesting that the DPP4 inhibition or the GLP-1R stimulation may have applications for treating inflammatory diseases.

The Soluble VEGF Receptor sFlt-1 Contributes to Impaired Neovascularization in Aged Mice.

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.

Stimulation of Alpha7 Nicotinic Acetylcholine Receptor Attenuates Nicotine-Induced Upregulation of MMP, MCP-1, and RANTES through Modulating ERK1/2/AP-1 Signaling Pathway in RAW264.7 and MOVAS Cells.

Vagus nerve stimulation through alpha7 nicotine acetylcholine receptors (α7-nAChR) signaling had been demonstrated attenuation of inflammation. This study aimed to determine whether PNU-282987, a selective α7-nAChR agonist, affected activities of matrix metalloproteinase (MMP) and inflammatory cytokines in nicotine-treatment RAW264.7 and MOVAS cells and to assess the underlying molecular mechanisms. RAW264.7 and MOVAS cells were treated with nicotine at different concentrations (0, 1, 10, and 100 ng/ml) for 0-120 min. Nicotine markedly stimulated the phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2) and c-Jun in RAW264.7 cells. Pretreatment with U0126 significantly suppressed phosphorylation of ERK1/2 and further attenuated nicotine-induced activation of c-Jun and upregulation of MMP-2, MMP-9, monocyte chemotactic protein- (MCP-) 1, and regulated upon activation normal T cell expressed and secreted (RANTES). Similarly, nicotine treatment also increased phosphorylation of c-Jun and expressions of MMP-2, MMP-9, MCP-1, and RANTES in MOVAS cells. When cells were pretreated with PNU-282987, nicotine-induced activations of ERK1/2 and c-Jun in RAW264.7 cells and c-Jun in MOVAS cells were effectively inhibited. Furthermore, nicotine-induced secretions of MMP-2, MMP-9, MCP-1, and RANTES were remarkably downregulated. Treatment with α7-nAChR agonist inhibits nicotine-induced upregulation of MMP and inflammatory cytokines through modulating ERK1/2/AP-1 signaling in RAW264.7 cells and AP-1 in MOVAS cells, providing a new therapeutic for abdominal aortic aneurysm.

Exercise restores muscle stem cell mobilization, regenerative capacity and muscle metabolic alterations via adiponectin/AdipoR1 activation in SAMP10 mice.

BACKGROUND: Exercise train (ET) stimulates muscle response in pathological conditions, including aging. The molecular mechanisms by which exercise improves impaired adiponectin/adiponectin receptor 1 (AdipoR1)-related muscle actions associated with aging are poorly understood. Here we observed that in a senescence-accelerated mouse prone 10 (SAMP10) model, long-term ET modulated muscle-regenerative actions. METHODS: 25-week-old male SAMP10 mice were randomly assigned to the control and the ET (45 min/time, 3/week) groups for 4 months. Mice that were maintained in a sedentary condition served controls. RESULTS: ET ameliorated aging-related muscle changes in microstructure, mitochondria, and performance. The amounts of proteins or mRNAs for p-AMPKα, p-Akt, p-ERK1/2, p-mTOR, Bcl-XL, p-FoxO3, peroxisome proliferators-activated receptor-γ coactivator, adiponectin receptor1 (adpoR1), and cytochrome c oxidase-IV, and the numbers of CD34+ /integrin-α7+ muscle stem cells (MuSCs) and proliferating cells in the muscles and bone-marrow were enhanced by ET, whereas the levels of p-GSK-3α and gp91phox proteins and apoptotic cells were reduced by ET. The ET also resulted in increased levels of plasma adiponectin and the numbers of bone-marrow (BM)-derived circulating CD34+ /integrin-α7+ MuSCs and their functions. Integrin-α7+ MuSCs of exercised mice had improved changes of those beneficial molecules. These ET-mediated aged muscle benefits were diminished by adiponectin and AdipoR1 blocking as well as AMPK inhibition. Finally, recombinant mouse adiponectin enhanced AMPK and mTOR phosphorylations in BM-derived integrin-α7+ cells. CONCLUSIONS: These findings suggest that ET can improve aging-related impairments of BM-derived MuSC regenerative capacity and muscle metabolic alterations via an AMPK-dependent mechanism that is mediated by an adiponectin/AdipoR1 axis in SAMP10 mice.

Dipeptidyl peptidase- IV inhibitor alogliptin improves stress-induced insulin resistance and prothrombotic state in a murine model.

BACKGROUND: Stress evokes lipolytic release of free fatty acid (FFA) and low-grade inflammation in visceral adipose tissue, mediated by increased adipokine secretion, and contributes to glucose metabolism disorder and prothrombotic state. We tested the hypothesis that alogliptin, a dipeptidyl peptidase-4 inhibitor, can ameliorate the biological effects of chronic stress in mice. METHOD AND RESULTS: C57BL/6J mice were subjected to 2-week intermittent restraint stress and orally treated with vehicle or alogliptin (dose: 15 or 45mg/kg/day). Plasma levels of lipids, proinflammatory cytokines (monocyte chemoattractant protein-1, tumor necrosis factor-α, and interleukin-6), and 8-hydroxydeoxyguanosine were measured with enzyme-linked immunosorbent assay. Monocyte/macrophage accumulation in inguinal white adipose tissue (WAT) was examined by CD11b-positive cell count and mRNA expression of CD68 and F4/80 was examined by immunohistochemistry and RT-PCR, respectively. The mRNA levels of the above-mentioned proinflammatory cytokines, NADPH oxidase 4, adiponectin, and coagulation factors (plasminogen activation inhibitor-1 and tissue factor) in WAT were also assessed with RT-PCR. Glucose metabolism was assessed by glucose and insulin tolerance tests, plasma levels of DPP-4 activity, glucagon-like peptide-1, expression of DPP-4, insulin receptor substrate-1 and glucose transporter 4 in WAT and skeletal muscle. Alogliptin administration suppressed stress-induced FFA release, oxidative stress, adipose tissue inflammation, DPP-4 activation, and prothrombotic state in a dose-dependent manner, and improved insulin sensitivity in stressed mice. CONCLUSIONS: The results indicate that alogliptin improves stress-induced prothrombotic state and insulin resistance; suggesting that alogliptin could have beneficial therapeutic effects against cardiovascular complications in diabetic patients under stress.

Cathepsin S Activity Controls Injury-Related Vascular Repair in Mice via the TLR2-Mediated p38MAPK and PI3K-Akt/p-HDAC6 Signaling Pathway.

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.

Impact of circulating cathepsin K on the coronary calcification and the clinical outcome in chronic kidney disease patients.

Chronic kidney disease (CKD) is a cause of coronary artery calcification (CAC) and an independent predictor of major adverse cardiac and cerebrovascular events (MACCE). Cathepsin K (CatK) is a lysosomal cysteine protease which affects vascular calcification and glucose metabolism disorder. We investigated the relationships among CatK, CAC, diabetes mellitus (DM) and MACCE in CKD patients. 113 consecutive CKD patients were enrolled. Their CAC was evaluated by computed tomography. Their plasma CatK level was measured by ELISA. They were divided into two groups by CatK levels and followed up for up to 3 years. The impact of CatK was analyzed in all participants, diabetic patients and non-diabetic patients. Kaplan-Meier analysis demonstrated a significant higher incidence of MACCE in the high CatK group (P = 0.028). The CatK level was significantly higher in patients with MACCE compared to that in patients without MACCE (P = 0.034). Cox's model revealed the higher plasma CatK and BNP level as independent predictors of MACCE (P = 0.043 and P < 0.01, respectively). Only in non-diabetic patients, there was a significant correlation between CatK and CAC score, and high CatK group had a significant higher level of LDL-C and LDL-C/HDL-C ratio (P < 0.05 and P < 0.001, respectively) than low CatK group. And these lipid disorders were independent predictors of CatK elevation. In CKD patients, our results indicated an impact of higher CatK level on their MACCE. The significant association among the CatK level, CAC and MACCE was found in non-diabetic CKD patients.

Cathepsin S activity controls ischemia-induced neovascularization in mice.

BACKGROUND: Evidence from human and animal studies has demonstrated elevated levels of the cysteine protease cathepsin S (CatS) in hypoxic atherosclerotic lesions. We hypothesized that silencing of CatS gene would suppress ischemia-induced angiogenic action. METHODS AND RESULTS: Left femoral artery ligation-induced ischemia in mice showed the increased expression and activity of CatS in the ischemic muscle. The CatS-deficiency (CatS(-/-)) mice showed impaired functional recovery following hindlimb ischemia and reduced levels of peroxisome proliferator-activated receptor-γ (PPAR-γ), phospho-Akt (p-Akt), p-endothelial nitric oxide synthase, p-extracellular signal-regulated kinase1/2 (Erk1/2), p-p38 mitogen-activated protein kinase, and vascular endothelial growth factor (VEGF) proteins, as well as reduced levels of matrix metalloproteinase-9 and macrophage infiltration in the ischemic muscles. In vitro, CatS silencing reduced the levels of these targeted essential molecules for angiogenesis and vasculogenesis. Together, the results indicated that the effects of CatS knockdown led to defective endothelial cell invasion, proliferation, and tube formation. This notion was reinforced by the finding that CatS inhibition led to a decreased PPAR-γ level and VEGF/Erk1/2 signaling activation in response to ischemia. CatS(-/-) resulted in decreased circulating EPC-like CD31(+)/c-Kit(+) cells, accompanied by the reduction of the cellular levels of PPAR-γ, p-Akt, and VEGF induced by ischemic stress. Transplantation of bone-marrow-derived mononuclear cells from CatS(+/+) mice restored neovascularization in CatS(-/-) mice. CONCLUSIONS: CatS activity controls ischemia-induced neovascularization partially via the modulation of PPAR-γ and VEGF/Akt signaling activation.

Renin inhibition reduces atherosclerotic plaque neovessel formation and regresses advanced atherosclerotic plaques.

OBJECTIVE: The interaction between the renin-angiotensin system and toll-like receptors (TLRs) in the pathogenesis of advanced atherosclerotic plaques is not well understood. We studied the effects of the renin inhibitor aliskiren on the progression of advanced atherosclerotic plaque in apolipoprotein E-deficient (ApoE(-/-)) mice with a special focus on plaque neovessel formation. METHODS AND RESULTS: Four-wk-old ApoE(-/-) mice were fed a high-fat diet for 8 wks, and the mice were randomly assigned to one of three groups and administered a vehicle, hydralazine, or aliskiren for an additional 12 wks. Aliskiren reduced the atherosclerotic plaque area and plaque neovessel density. It increased the plaque collagen and elastin contents, and reduced plasma angiotensin II levels and plaque macrophage infiltration and cathepsin S (CatS) protein. Aliskiren also decreased the levels of AT1R, gp91phox, TLR2, monocyte chemotactic protein-1, and CatS mRNAs in the aortic roots. Hydralazine had no beneficial vascular effects, although its administration resulted in the same degree of blood pressure reduction as aliskiren. CatS deficiency mimicked the aliskiren-mediated vasculoprotective effect in the ApoE(-/-) mice, but aliskiren showed no further benefits in ApoE(-/-) CatS(-/-) mice. In vitro, TLR2 silencing reduced CatS expression induced by angiotensin II. Moreover, aliskiren or the inhibition of CatS impaired the endothelial cell angiogenic action in vitro or/and ex vivo. CONCLUSION: Renin inhibition appears to inhibit advanced plaque neovessel formation in ApoE(-/-) mice and to decrease the vascular inflammatory action and extracellular matrix degradation, partly by reducing AT1R/TLR2-mediated CatS activation and activity, thus regressing advanced atherosclerosis.