Cathepsin K deficiency prevented stress-related thrombosis in a mouse FeCl3 model.

BACKGROUND: Exposure to chronic psychological stress (CPS) is a risk factor for thrombotic cardiocerebrovascular diseases (CCVDs). The expression and activity of the cysteine cathepsin K (CTSK) are upregulated in stressed cardiovascular tissues, and we investigated whether CTSK is involved in chronic stress-related thrombosis, focusing on stress serum-induced endothelial apoptosis. METHODS AND RESULTS: Eight-week-old wild-type male mice (CTSK+/+) randomly divided to non-stress and 3-week restraint stress groups received a left carotid artery iron chloride3 (FeCl3)-induced thrombosis injury for biological and morphological evaluations at specific timepoints. On day 21 post-stress/injury, the stress had enhanced the arterial thrombi weights and lengths, in addition to harmful alterations of plasma ADAMTS13, von Willebrand factor, and plasminogen activation inhibitor-1, plus injured-artery endothelial loss and CTSK protein/mRNA expression. The stressed CTSK+/+ mice had increased levels of injured arterial cleaved Notch1, Hes1, cleaved caspase8, matrix metalloproteinase-9/-2, angiotensin type 1 receptor, galactin3, p16IN4A, p22phox, gp91phox, intracellular adhesion molecule-1, TNF-α, MCP-1, and TLR-4 proteins and/or genes. Pharmacological and genetic inhibitions of CTSK ameliorated the stress-induced thrombus formation and the observed molecular and morphological changes. In cultured HUVECs, CTSK overexpression and silencing respectively increased and mitigated stressed-serum- and H2O2-induced apoptosis associated with apoptosis-related protein changes. Recombinant human CTSK degraded γ-secretase substrate in a dose-dependent manor and activated Notch1 and Hes1 expression upregulation. CONCLUSIONS: CTSK appeared to contribute to stress-related thrombosis in mice subjected to FeCl3 stress, possibly via the modulation of vascular inflammation, oxidative production and apoptosis, suggesting that CTSK could be an effective therapeutic target for CPS-related thrombotic events in patients with CCVDs.

Dipeptidyl peptidase-4 disturbs adipocyte differentiation via the negative regulation of the glucagon-like peptide-1/adiponectin-cathepsin K axis in mice under chronic stress conditions.

Exposure to chronic psychosocial stress is a risk factor for metabolic disorders. Because dipeptidyl peptidase-4 (DPP4) and cysteinyl cathepsin K (CTSK) play important roles in human pathobiology, we investigated the role(s) of DPP4 in stress-related adipocyte differentiation, with a focus on the glucagon-like peptide-1 (GLP-1)/adiponectin-CTSK axis in vivo and in vitro. Plasma and inguinal adipose tissue from non-stress wild-type (DPP4+/+), DPP4-knockout (DPP4-/-) and CTSK-knockout (CTSK-/-) mice, and stressed DPP4+/+, DPP4-/-, CTSK-/-, and DPP4+/+ mice underwent stress exposure plus GLP-1 receptor agonist exenatide loading for 2 weeks and then were analyzed for stress-related biological and/or morphological alterations. On day 14 under chronic stress, stress decreased the weights of adipose tissue and resulted in harmful changes in the plasma levels of DPP4, GLP-1, CTSK, adiponectin, and tumor necrosis factor-α proteins and the adipose tissue levels of CTSK, preadipocyte factor-1, fatty acid binding protein-4, CCAAT/enhancer binding protein-α, GLP-1 receptor, peroxisome proliferator-activated receptor-γ, perilipin2, secreted frizzled-related protein-4, Wnt5α, Wnt11 and ß-catenin proteins and/or mRNAs as well as macrophage infiltration in adipose tissue; these changes were rectified by DPP4 deletion. GLP-1 receptor activation and CTSK deletion mimic the adipose benefits of DPP4 deficiency. In vitro, CTSK silencing and overexpression respectively prevented and facilitated stress serum and oxidative stress-induced adipocyte differentiation accompanied with changes in the levels of pref-1, C/EBP-α, and PPAR-γ in 3T3-L1 cells. Thus, these findings indicated that increased DPP4 plays an essential role in stress-related adipocyte differentiation, possibly through a negative regulation of GLP-1/adiponectin-CTSK axis activation in mice under chronic stress conditions.

Cathepsin S activity controls chronic stress-induced muscle atrophy and dysfunction in mice.

Exposure to chronic psychological stress (CPS) is an intractable risk factor for inflammatory and metabolic diseases. Lysosomal cysteinyl cathepsins play an important role in human pathobiology. Given that cathepsin S (CTSS) is upregulated in the stressed vascular and adipose tissues, we investigated whether CTSS participates in chronic stress-induced skeletal muscle mass loss and dysfunction, with a special focus on muscle protein metabolic imbalance and apoptosis. Eight-week-old male wildtype (CTSS+/+) and CTSS-knockout (CTSS-/-) mice were randomly assigned to non-stress and variable-stress groups. CTSS+/+ stressed mice showed significant losses of muscle mass, dysfunction, and fiber area, plus significant mitochondrial damage. In this setting, stressed muscle in CTSS+/+ mice presented harmful alterations in the levels of insulin receptor substrate 2 protein content (IRS-2), phospho-phosphatidylinositol 3-kinase, phospho-protein kinase B, and phospho-mammalian target of rapamycin, forkhead box-1, muscle RING-finger protein-1 protein, mitochondrial biogenesis-related peroxisome proliferator-activated receptor-γ coactivator-α, and apoptosis-related B-cell lymphoma 2 and cleaved caspase-3; these alterations were prevented by CTSS deletion. Pharmacological CTSS inhibition mimics its genetic deficiency-mediated muscle benefits. In C2C12 cells, CTSS silencing prevented stressed serum- and oxidative stress-induced IRS-2 protein reduction, loss of the myotube myosin heavy chain content, and apoptosis accompanied by a rectification of investigated molecular harmful changes; these changes were accelerated by CTSS overexpression. These findings demonstrated that CTSS plays a role in IRS-2-related protein anabolism and catabolism and cell apoptosis in stress-induced muscle wasting, suggesting a novel therapeutic strategy for the control of chronic stress-related muscle disease in mice under our experimental conditions by regulating CTSS activity.

Cathepsin S deficiency improves muscle mass loss and dysfunction via the modulation of protein metabolism in mice under pathological stress conditions.

Cathepsin S (CTSS) is a widely expressed cysteinyl protease that has garnered attention because of its enzymatic and non-enzymatic functions under inflammatory and metabolic pathological conditions. Here, we examined whether CTSS participates in stress-related skeletal muscle mass loss and dysfunction, focusing on protein metabolic imbalance. Eight-week-old male wildtype (CTSS+/+ ) and CTSS-knockout (CTSS-/- ) mice were randomly assigned to non-stress and variable-stress groups for 2 weeks, and then processed for morphological and biochemical studies. Compared with non-stressed mice, stressed CTSS+/+ mice showed significant losses of muscle mass, muscle function, and muscle fiber area. In this setting, the stress-induced harmful changes in the levels of oxidative stress-related (gp91phox and p22phox ,), inflammation-related (SDF-1, CXCR4, IL-1ß, TNF-α, MCP-1, ICAM-1, and VCAM-1), mitochondrial biogenesis-related (PPAR-γ and PGC-1α) genes and/or proteins and protein metabolism-related (p-PI3K, p-Akt, p-FoxO3α, MuRF-1, and MAFbx1) proteins; and these alterations were rectified by CTSS deletion. Metabolomic analysis revealed that stressed CTSS-/- mice exhibited a significant improvement in the levels of glutamine metabolism pathway products. Thus, these findings indicated that CTSS can control chronic stress-related skeletal muscle atrophy and dysfunction by modulating protein metabolic imbalance, and thus CTSS was suggested to be a promising new therapeutic target for chronic stress-related muscular diseases.

CTSS Modulates Stress-Related Carotid Artery Thrombosis in a Mouse FeCl3 Model.

BACKGROUND: Exposure to chronic psychological stress is a risk factor for metabolic cardiovascular disease. Given the important role of lysosomal CTSS (cathepsin S) in human pathobiology, we examined the role of CTSS in stress-related thrombosis, focusing on inflammation, oxidative stress, and apoptosis. METHODS: Six-week-old wild-type mice (CTSS+/+) and CTSS-deficient mice (CTSS-/-) randomly assigned to nonstress and 2-week immobilization stress groups underwent iron chloride3 (FeCl3)-induced carotid thrombosis surgery for morphological and biochemical studies. RESULTS: On day 14 poststress/surgery, stress had increased the lengths and weights of thrombi in the CTSS+/+ mice, plus harmful changes in the levels of PAI-1 (plasminogen activation inhibitor-1), ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 13 motifs), and vWF (von Willebrand factor) and arterial tissue CTSS expression. Compared to the nonstressed CTSS+/+ mice, the stressed CTSS-/- mice had decreased levels of PAI-1, vWF, TNF (tumor necrosis factor)-α, interleukin-1ß, toll-like receptor-4, cleaved-caspase 3, cytochrome c, p16INK4A, gp91phox, p22phox, ICAM-1 (intercellular adhesion molecule-1), MCP-1 (monocyte chemoattractant protein-1), MyD88 (myeloid differentiation primary response 88), and MMP (matrix metalloproteinase)-2/-9 and increased levels of ADAMTS13, SOD (superoxide dismutase)-1/-2, eNOS (endothelial NO synthase), p-Akt (phospho-protein kinase B), Bcl-2 (B-cell lymphoma-2), p-GSK3α/ß (phospho-glycogen synthase kinases alpha and beta), and p-Erk1/2 (phospho-extracellular signal-regulated kinase 1 and 2) mRNAs and/or proteins. CTSS deletion also reduced the arterial thrombus area and endothelial loss. A pharmacological inhibition of CTSS exerted a vasculoprotective action. In vitro, CTSS silencing and overexpression, respectively, reduced and increased the stressed serum and oxidative stress-induced apoptosis of human umbilical vein endothelial cells, and they altered apoptosis-related proteins. CONCLUSIONS: CTSS inhibition appeared to improve the stress-related thrombosis in mice that underwent FeCl3-induction surgery, possibly by reducing vascular inflammation, oxidative stress, and apoptosis. CTSS could thus become a candidate therapeutic target for chronic psychological stress-related thrombotic events in metabolic cardiovascular disease.

Overview of multifunctional cysteinyl cathepsins in atherosclerosis-based cardiovascular disease: from insights into molecular functions to clinical implications.

Cysteinyl cathepsins (CTSs) are widely known to have a proteolysis function that mediates recycling of unwanted proteins in endosomes and lysosomes, and investigation of CTSs has greatly improved with advances in live-imaging techniques both in vivo and in vitro, leading to three key findings. (1) CTSs are relocated from the lysosomes to other cellular spaces (i.e., cytosol, nucleus, nuclear membrane, plasma membrane, and extracellular milieu). (2) In addition to acidic cellular compartments, CTSs also exert biological activity in neutral environments. (3) CTSs also exert multiple nontraditional functions in, for example, extracellular matrix metabolism, cell signaling transduction, protein processing/trafficking, and cellular events. Various stimuli regulate the expression and activities of CTSs in vivo and vitro-e.g., inflammatory cytokines, oxidative stress, neurohormones, and growth factors. Accumulating evidence has confirmed the participation of CTSs in vascular diseases characterized by atherosclerosis, plaque rupture, thrombosis, calcification, aneurysm, restenosis/in-stent-restenosis, and neovasel formation. Circulating and tissue CTSs are promising as biomarkers and as a diagnostic imaging tool in patients with atherosclerosis-based cardiovascular disease (ACVD), and pharmacological interventions with their specific and non-specific inhibitors, and cardiovascular drugs might have potential for the therapeutic targeting of CTSs in animals. This review focuses on the update findings on CTS biology and the involvement of CTSs in the initiation and progression of ACVD and discusses the potential use of CTSs as biomarkers and small-molecule targets to prevent deleterious nontraditional functions in ACVD.

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.

Human umbilical cord-derived mesenchymal stromal cells ameliorate aging-associated skeletal muscle atrophy and dysfunction by modulating apoptosis and mitochondrial damage in SAMP10 mice.

BACKGROUND: Skeletal muscle mass and function losses in aging individuals are associated with quality of life deterioration and disability. Mesenchymal stromal cells exert immunomodulatory and anti-inflammatory effects and could yield beneficial effects in aging-related degenerative disease. METHODS AND RESULTS: We investigated the efficacy of umbilical cord-derived mesenchymal stromal cells (UC-MSCs) on sarcopenia-related skeletal muscle atrophy and dysfunction in senescence-accelerated mouse prone 10 (SAMP10) mice. We randomly assigned 24-week-old male SAMP10 mice to a UC-MSC treatment group and control group. At 12 weeks post-injection, the UC-MSC treatment had ameliorated sarcopenia-related muscle changes in performance, morphological structures, and mitochondria biogenesis, and it enhanced the amounts of proteins or mRNAs for myosin heavy chain, phospho-AMP-activated protein kinase, phospho-mammalian target of rapamycin, phospho-extracellular signal-regulated kinase1/2, peroxisome proliferator-activated receptor-γ coactivator, GLUT-4, COX-IV, and hepatocyte growth factor in both gastrocnemius and soleus muscles, and it reduced the levels of proteins or mRNAs for cathepsin K, cleaved caspase-3/-8, tumor necrosis factor-α, monocyte chemoattractant protein-1, and gp91phox mRNAs. The UC-MSC treatment retarded mitochondria damage, cell apoptosis, and macrophage infiltrations, and it enhanced desmin/laminin expression and proliferating and CD34+/Integrin α7+ cells in both types of skeletal muscle of the SAMP10 mice. In vitro, we observed increased levels of HGF, PAX-7, and MoyD mRNAs at the 4th passage of UC-MSCs. CONCLUSIONS: Our results suggest that UC-MSCs can improve sarcopenia-related skeletal muscle atrophy and dysfunction via anti-apoptosis, anti-inflammatory, and mitochondrial biogenesis mechanisms that might be mediated by an AMPK-PGC1-α axis, indicating that UC-MSCs may provide a promising treatment for sarcopenia/muscle diseases.

Cathepsin K Deficiency Prevented Kidney Damage and Dysfunction in Response to 5/6 Nephrectomy Injury in Mice With or Without Chronic Stress.

BACKGROUND: Chronic psychological stress is a risk factor for kidney disease, including kidney dysfunction and hypertension. Lysosomal CatK (cathepsin K) participates in various human pathobiologies. We investigated the role of CatK in kidney remodeling and hypertension in response to 5/6 nephrectomy injury in mice with or without chronic stress. METHODS: Male 7-week-old WT (wild type; CatK+/+) and CatK-deficient (CatK-/-) mice that were or were not subjected to chronic stress underwent 5/6 nephrectomy. At 8 weeks post-stress/surgery, the stress was observed to have accelerated injury-induced glomerulosclerosis, proteinuria, and blood pressure elevation. RESULTS: Compared with the nonstressed mice, the stressed mice showed increased levels of TLR (Toll-like receptor)-2/4, p22phox, gp91phox, CatK, MMP (matrix metalloproteinase)-2/9, collagen type I and III genes, PPAR-γ (peroxisome proliferator-activated receptor-gamma), NLRP-3 (NOD-like receptor thermal protein domain associated protein 3), p21, p16, and cleaved caspase-8 proteins, podocyte foot process effacement, macrophage accumulation, apoptosis, and decreased levels of Bcl-2 (B cell lymphoma 2) and Sirt1, as well as decreased glomerular desmin expression in the kidneys. These harmful changes were retarded by the genetic or pharmacological inhibition of CatK. Consistently, CatK inhibition ameliorated 5/6 nephrectomy-related kidney injury and dysfunction. In mesangial cells, CatK silencing or overexpression, respectively, reduced or increased the PPAR-γ and cleaved caspase-8 protein levels, providing evidence and a mechanistic explanation of CatK's involvement in PPAR-γ/caspase-8-mediated cell apoptosis in response to superoxide and stressed serum. CONCLUSIONS: These results demonstrate that CatK plays an essential role in kidney remodeling and hypertension in response to 5/6 nephrectomy or stress, possibly via a reduction of glomerular inflammation, apoptosis, and fibrosis, suggesting a novel therapeutic strategy for controlling kidney injury in mice under chronic psychological stress conditions.

Cathepsin K activity controls cachexia-induced muscle atrophy via the modulation of IRS1 ubiquitination.

BACKGROUND: Cachexia is a complicated metabolic disorder that is characterize by progressive atrophy of skeletal muscle. Cathepsin K (CTSK) is a widely expressed cysteine protease that has garnered attention because of its enzymatic and non-enzymatic functions in signalling in various pathological conditions. Here, we examined whether CTSK participates in cancer-induced skeletal muscle loss and dysfunction, focusing on protein metabolic imbalance. METHODS: Male 9-week-old wild-type (CTSK+/+ , n = 10) and CTSK-knockout (CTSK-/- , n = 10) mice were injected subcutaneously with Lewis lung carcinoma cells (LLC; 5 × 105 ) or saline, respectively. The mice were then subjected to muscle mass and muscle function measurements. HE staining, immunostaining, quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and western blotting were used to explore the CTSK expression and IRS1/Akt pathway in the gastrocnemius muscle at various time points. In vitro measurements included CTSK expression, IRS1/Akt pathway-related target molecule expressions, and the diameter of C2C12 myotubes with or without LLC-conditioned medium (LCM). An IRS1 ubiquitin assay, and truncation, co-immunoprecipitation, and co-localization experiments were also performed. RESULTS: CTSK+/+ cachectic animals exhibited loss of skeletal muscle mass (muscle weight loss of 15%, n = 10, P < 0.01), muscle dysfunction (grip strength loss > 15%, n = 10, P < 0.01), and fibre area (average area reduction > 30%, n = 5, P < 0.01). Compared with that of non-cachectic CTSK+/+ mice, the skeletal muscle of cachectic CTSK+/+ mice exhibited greater degradation of insulin receptor substrate 1 (IRS1, P < 0.01). In this setting, cachectic muscles exhibited decreases in the phosphorylation levels of protein kinase B (Akt308 , P < 0.01; Akt473 , P < 0.05) and anabolic-related proteins (the mammalian target of rapamycin, P < 0.01) and increased levels of catabolism-related proteins (muscle RING-finger protein-1, P < 0.01; MAFbx1, P < 0.01) in CTSK+/+ mice (n = 3). Although there was no difference in LLC tumour growth (n = 10, P = 0.44), CTSK deletion mitigated the IRS1 degradation, loss of the skeletal muscle mass (n = 10, P < 0.01), and dysfunction (n = 10, P < 0.01). In vitro, CTSK silencing prevented the IRS1 ubiquitination and loss of the myotube myosin heavy chain content (P < 0.01) induced by LCM, and these changes were accelerated by CTSK overexpression even without LCM. Immunoprecipitation showed that CTSK selectively acted on IRS1 in the region of amino acids 268 to 574. The results of co-transfection of IRS1-N-FLAG or IRS1-C-FLAG with CTSK suggested that CTSK selectively cleaves IRS1 and causes ubiquitination-related degradation of IRS1. CONCLUSIONS: These results demonstrate that CTSK plays a novel role in IRS1 ubiquitination in LLC-induced muscle wasting, and suggest that CTSK could be an effective therapeutic target for cancer-related cachexia.

Granulocyte colony-stimulating factor protected against brain injury in a rat cerebral hemorrhage model by modulating inflammation.

Granulocyte colony-stimulating factor (G-CSF) has been reported to exert a protective effect against secondary brain damage, but the underlying mechanisms remain unknown. We explored the ability of G-CSF to protect the brain from injury in a rat autologous blood-induced model of intracerebral hemorrhage (ICH), with a special focus on the anti-inflammation effect. An ICH was induced in 8-week-old male rats by an infusion of autologous blood, and the rats were then randomly assigned to five treatment groups: sham, ICH, and ICH+ low-dose (25 µg/kg), middle-dose (50 µg/kg), and high-dose (75 µg/kg) G-CSF. We then evaluated the levels of brain inflammation-related genes and proteins. The levels of tumor-necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) mRNA increased between days 1 and 14 post-ICH, with the highest expression on day 3. These changes were rectified by G-CSF in a dose-dependent manner. At day 3 post-injury, an elevation of the nuclear factor-kappa B (NF-κB) p65 protein level and a reduction of the inhibitor of NF-κB alpha (IκBα) protein level were observed; G-CSF treatment exerted a beneficial effect on both protein expressions. The expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins were increased; these changes were rectified by the highest dose of G-CSF. The brain-protecting effects of G-CSF are likely to be attributable, at least in part, to attenuation of the TNF-α, IL-6, iNOS, and COX-2 expressions induced by NF-κB activation in the brain tissues of this autologous blood-induced ICH rat model.

The sGC-cGMP Signaling Pathway as a Potential Therapeutic Target in Doxorubicin-Induced Heart Failure: A Narrative Review.

The anti-cancer agent doxorubicin (DOX) has high cardiotoxicity that is linked to DOX-mediated increase in oxidative stress, mitochondrial iron overload, DNA damage, autophagy, necrosis, and apoptosis, all of which are also associated with secondary tumorigenicity. This limits the clinical application of DOX therapies. Previous studies have attributed DOX-mediated cardiotoxicity to mitochondrial iron accumulation and the production of reactive oxygen species (ROS), which seem to be independent of its anti-tumor DNA damaging effects. Chemo-sensitization of soluble guanylate cyclase (sGC) in the cyclic guanosine monophosphate (cGMP) pathway induces tumor cell death despite the cardiotoxicity associated with DOX treatment. However, sGC-cGMP signaling must be activated during heart failure to facilitate myocardial cell survival. The sGC pathway is dependent on nitric oxide and signal transduction via the nitric oxide-sGC-cGMP pathway and is attenuated in various cardiovascular diseases. Additionally, cGMP signaling is regulated by the action of certain phosphodiesterases (PDEs) that protect the heart by inhibiting PDE, an enzyme that hydrolyses cGMP to GMP activity. In this review, we discuss the studies describing the interactions between cGMP regulation and DOX-mediated cardiotoxicity and their application in improving DOX therapeutic outcomes. The results provide novel avenues for the reduction of DOX-induced secondary tumorigenicity and improve cellular autonomy during DOX-mediated cardiotoxicity.

Proliferin-1 Ameliorates Cardiotoxin-Related Skeletal Muscle Repair in Mice.

BACKGROUND: We recently demonstrated that proliferin-1 (PLF-1) functions as an apoptotic cell-derived growth factor and plays an important role in vascular pathobiology. We therefore investigated its role in muscle regeneration in response to cardiotoxin injury. METHODS AND RESULTS: To determine the effects of PLF-1 on muscle regeneration, we used a CTX-induced skeletal muscle injury model in 9-week-old male mice that were administered with the recombinant PLF-1 (rPLF-1) or neutralizing PLF-1 antibody. The injured muscles exhibited increased levels of PLF-1 gene expression in a time-dependent manner. On day 14 after injury, rPLF-1 supplementation ameliorated CTX-induced alterations in muscle fiber size, interstitial fibrosis, muscle regeneration capacity, and muscle performance. On day 3 postinjury, rPLF-1 increased the levels of proteins or genes for p-Akt, p-mTOR, p-GSK3α/ß, p-Erk1/2, p-p38MAPK, interleukin-10, Pax7, MyoD, and Cyclin B1, and it increased the numbers of CD34+/integrin-α7+ muscle stem cells and proliferating cells in the muscles and/or bone marrow of CTX mice. An enzyme-linked immunosorbent assay revealed that rPLF-1 suppressed the levels of plasma tumor necrosis factor-α and interleukin-1ß in CTX mice. PLF-1 blocking accelerated CTX-related muscle damage and dysfunction. In C2C12 myoblasts, rPLF-1 increased the levels of proteins for p-Akt, p-mTOR, p-GSK3α/ß, p-Erk1/2, and p-p38MAPK as well as cellular functions; and these effects were diminished by the depletion of PLF-1 or silencing of its mannose-6-phosphate receptor. CONCLUSIONS: These findings demonstrated that PLF-1 can improve skeletal muscle repair in response to injury, possibly via the modulation of inflammation and proliferation and regeneration, suggesting a novel therapeutic strategy for the management of skeletal muscle diseases.

Dipeptidyl peptidase-4 inhibition prevents lung injury in mice under chronic stress via the modulation of oxidative stress and inflammation.

Exposure to chronic psychosocial stress is a risk factor for various pulmonary diseases. In view of the essential role of dipeptidyl peptidase 4 (DPP4) in animal and human lung pathobiology, we investigated the role of DPP4 in stress-related lung injury in mice. Eight-week-old male mice were randomly divided into a non-stress group and a 2-week immobilization stress group. Non-stress control mice were left undisturbed. The mice subjected to immobilized stress were randomly assigned to the vehicle or the DPP4 inhibitor anagliptin for 2 weeks. Chronic stress reduced subcutaneous and inguinal adipose volumes and increased blood DPP4 levels. The stressed mice showed increased levels in the lungs of genes and/or proteins related to oxidative stress (p67phox, p47phox, p22phox and gp91phox), inflammation (monocyte chemoattractant protein-1, vascular cell adhesion molecule-1, and intracellular adhesion molecule-1), apoptosis (caspase-3, -8, -9), senescence (p16INK4A, p21, and p53) and proteolysis (matrix metalloproteinase-2 to -9, cathepsin S/K, and tissue inhibitor of matrix metalloproteinase-1 and -2), and reduced levels of eNOS, Sirt1, and Bcl-2 proteins; and these effects were reversed by genetic and pharmacological inhibitions of DPP4. We then exposed human umbilical vein endothelial cells in vitro to hydrogen peroxide; anagliptin treatment was also observed to mitigate oxidative and inflammatory molecules in this setting. Anagliptin can improve lung injury in stressed mice, possibly by mitigating vascular inflammation, oxidative stress production, and proteolysis. DPP4 may become a new therapeutic target for chronic psychological stress-related lung disease in humans and animals.

Role of Dipeptidyl Peptidase-4 in Atherosclerotic Cardiovascular Disease in Humans and Animals with Chronic Stress.

Exposure to psychosocial stress is a risk factor for cardiovascular disease, including vascular atherosclerosis-based cardiovascular disease (ACVD). Dipeptidyl peptidase-4 (DPP-4) is a complex enzyme that acts as a membrane-anchored cell surface exopeptidase. DPP-4 is upregulated in metabolic and inflammatory cardiovascular disorders. DPP-4 exhibits many physiological and pharmacological functions by regulating its extremely abundant substrates, such as glucagon-like peptide-1 (GLP-1). Over the last 10 years, emerging data have demonstrated unexpected roles of DPP-4 in extracellular and intracellular signaling, immune activation, inflammation, oxidative stress production, cell apoptosis, insulin resistance, and lipid metabolism. This mini-review focuses on recent novel findings in this field, highlighting a DPP-4-mediated regulation of GLP-1-dependent and -independent signaling pathways as a potential therapeutic molecular target in treatments of chronic psychological stress-related ACVD in humans and animals.

Adaptive immune disorders in hypertension and heart failure: focusing on T-cell subset activation and clinical implications.

: Hypertension is a growing health concern worldwide. Established hypertension is a causative factor of heart failure, which is characterized by increased vascular resistance and intractable uncontrolled blood pressure. Hypertension and heart failure have multiple causes and complex pathophysiology but cellular immunity is thought to contribute to the development of both. Recent studies showed that T cells play critical roles in hypertension and heart failure in humans and animals, with various stimuli leading to the formation of effector T cells that infiltrate the cardiovascular wall. Monocytes/macrophages also accumulate in the cardiovascular wall. Various cytokines (e.g. interleukin-6, interleukin-17, interleukin-10, tumor necrosis factor-α, and interferon-γ) released from immune cells of various subtypes promote vascular senescence and elastic laminal degradation as well as cardiac fibrosis and/or hypertrophy, leading to cardiovascular structural alterations and dysfunction. Recent laboratory evidence has defined a link between inflammation and the immune system in initiation and progression of hypertension and heart failure. Moreover, cross-talk among natural killer cells, adaptive immune cells (T cells and B cells), and innate immune cells (i.e. monocytes, macrophages, neutrophils, and dendritic cells) contributes to end-cardiovasculature damage and dysfunction in hypertension and heart failure. Clinical and experimental studies on the diagnostic potential of T-cell subsets revealed that blood regulatory T cells, CD4 cells, CD8 T cells, and the ratio of CD4 to CD8 T cells show promise as biomarkers of hypertension and heart failure. Therapeutic interventions to suppress activation of these cells may prove beneficial in reducing end-organ damage and preventing consequences of cardiovascular failure, including hypertension of heart failure.

Cathepsin K Deficiency Impaired Ischemia-Induced Neovascularization in Aged Mice.

BACKGROUND: Aging is a major risk factor for cardiovascular disease. Cysteine protease cathepsin K (CatK) has been implicated in the process of angiogenesis, but the exact roles of individual CatK in vessel formation during aging are poorly understood. METHODS AND RESULTS: To study the putative role of CatK in ischemia-induced angiogenesis, we applied a hindlimb ischemia model to aged wild-type (CatK+/+) and CatK-deficient (CatK-/-) mice. A serial laser Doppler blood-flow analysis revealed that the recovery of the ischemic/normal blood-flow ratio in the aged CatK-/-mice was impaired throughout the follow-up period. On postoperative day 14, CatK deficiency had also impaired capillary formation. CatK deficiency reduced the levels of cleaved Notch1, phospho-Akt, and/or vascular endothelial growth factor (VEGF) proteins in the ischemic muscles and bone marrow-derived c-Kit+ cells. A flow cytometry analysis revealed that CatK deficiency reduced the numbers of endothelial progenitor cell (EPC)-like CD31+/c-Kit+ cells in the peripheral blood as well as the ischemic vasculature. In vitro experiments, CatK-/- impaired bone-derived c-Kit+ cellular functions (migration, invasion, proliferation, and tubulogenesis) in aged mice. Our findings demonstrated that aging impaired the ischemia-induced angiogenesis associated with the reductions of the production and mobilization of CD31+/c-Kit+ cells in mice. CONCLUSIONS: These findings established that the impairment of ischemia-induced neovascularization in aged CatK-/- mice is due, at least in part, to the reduction of EPC mobilization and the homing of the cells into vasculature that is associated with the impairment of Notch1 signaling activation at advanced ages.

Substance P enhances the local activation of NK1R-expressing c-kit+ cardiac progenitor cells in right atrium of ischemia/reperfusion-injured heart.

BACKGROUND: Localization of neurokinin 1 receptor (NK1R), the endogenous receptor for neuropeptide substance P (SP), has already been described for the right atrium (RA) of the heart. However, the biological role of SP/NK1R signal pathways in the RA remains unclear. Sprague-Dawley rats were randomly divided into 4 groups (n = 22 each); subjected to sham, ischemia/reperfusion-injury (I/R), I/R with 5 nmole/kg SP injection (SP + I/R), and SP + I/R with 1 mg/kg RP67580 injection (RP, a selective non-peptide tachykinin NK1R antagonist) (RP/SP + I/R). The left anterior descending coronary artery was occluded for 40 min followed by 1 day reperfusion with SP or SP + RP or without either. After 1 day, both atria and ventricles as well as the heart apexes were collected. RESULTS: SP promoted the expression of c-Kit, GATA4, Oct4, Nanog, and Sox2 in only the RA of the SP + I/R rats via NK1R activation. In agreement with these observations, NK1R-expressing c-Kit+ Nkx2.5+GATA4+ cardiac progenitor cells (CPCs) in the ex vivo RA explant outgrowth assay markedly migrated out from RA1 day SP + I/R approximately 2-fold increase more than RA1 day I/R. Treatment of SP promoted proliferation, migration, cardiosphere formation, and potential to differentiate into cardiomyocytes. Using RP inhibitor, NK1R antagonist not only inhibited cell proliferation and migration but also reduced the formation of cardiosphere and differentiation of c-Kit+ CPCs. CONCLUSION: SP/NK1R might play a role as a key mediator involved in the cellular response to c-Kit+ CPC expansion in RA of the heart within 24 h after I/R.

Increased dipeptidyl peptidase-4 accelerates chronic stress-related thrombosis in a mouse carotid artery model.

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.

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.