Haiying Jiang

Cathepsin K-mediated notch1 activation contributes to neovascularization in response to hypoxia

Cysteine proteases play important roles in pathobiology. Here we reveal that cathepsin K (CatK) has a role in ischaemia-induced neovascularization. Femoral artery ligation-induced ischaemia in mice increases CatK expression and activity, and CatK-deficient mice show impaired functional recovery following hindlimb ischaemia. CatK deficiency reduces the levels of cleaved Notch1 (c-Notch1), Hes1 Hey1, Hey2, vascular endothelial growth factor, Flt-1 and phospho-Akt proteins of the ischaemic muscles. In endothelial cells, silencing of CatK mimicked, whereas CatK overexpression enhanced, the levels of c-Notch1 and the expression of Notch downstream signalling molecules, suggesting CatK contributes to Notch1 processing and activates downstream signalling. Moreover, CatK knockdown leads to defective endothelial cell invasion, proliferation and tube formation, and CatK deficiency is associated with decreased circulating endothelial progenitor cells-like CD31(+)/c-Kit(+) cells in mice following hindlimb ischaemia. Transplantation of bone marrow-derived mononuclear cells from CatK(+/+) mice restores the impairment of neovascularization in CatK(-/-) mice. We conclude that CatK may be a potential therapeutic target for ischaemic disease.

Cathepsin K Activity Controls Injury-Related Vascular Repair in Mice

Cathepsin K (CatK) is one of the most potent mammalian collagenases. We showed previously the increased expression of CatK in human and animal atherosclerotic lesions. Here, we hypothesized that ablation of CatK mitigates injury-induced neointimal hyperplasia. Male wild-type (CatK+/+) and CatK-deficient (CatK−/−) mice underwent ligation or a combination of ligation and polyethylene cuff-replacement injuries to the right common carotid artery just proximal to its bifurcation, and they were then processed for morphological and biochemical studies at specific time points. On operative day 28, CatK−/− significantly reduced neointimal formation and neovessel formation in both single- and combination-injured arteries compared with the Cat K+/+ mice. At early time points, CatK−/− reduced the lesion macrophage contents and medial smooth muscle cell proliferation, the mRNA levels of monocyte chemoattractant protein-1, toll-like receptor-2, toll-like receptor-4, chemokine ligand-12, and the gelatinolytic activity related to matrix metalloproteinase-2/-9. An aorta-explant assay revealed that smooth muscle cell movement was impaired in the CatK−/− mice compared with the CatK+/+ mice. In addition, the smooth muscle cells and macrophages from CatK−/− mice had less invasive ability through a reconstituted basement membrane barrier. This vasculoprotective effect was mimicked by Cat inhibition with trans-epoxysuccinyl-L-leucylamido-{4-guanidino} butane (E64d). These results demonstrate an essential role of CatK in neointimal lesion formation in response to injury, possibly via the reduction of toll-like receptor-2/-4–mediated inflammation and smooth muscle cell proliferation, suggesting a novel therapeutic strategy for the control of endovascular treatment–related restenosis by regulating CatK activity.

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.

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.

Increased Plasma Dipeptidyl Peptidase-4 Activities in Patients with Coronary Artery Disease

Dipeptidyl peptidase-4 (DPP4) is one of the most potent mammalian serine proteases participated in the pathogenesis of subclinical atherosclerosis. Here we investigated whether the plasma soluble form of DPP4 is associated with the prevalence of coronary artery disease (CAD) with and without diabetes mellitus (DM). A cross-sectional study was conducted of 496 aged 26–81 years with (n = 362) and without (n = 134) CAD. Plasma DPP4 activity, high sensitive C-reactive protein (hs-CRP), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein levels were measured. The coronary atherosclerotic plaques were evaluated by coronary angiography. The CAD patients with (n = 84) and without (n = 278) DM had significantly higher DPP4 levels (11.8 ± 3.1 vs. 6.9 ± 3.5 ng/mL, P<0.01) than the nonCAD subjects. The acute coronary syndrome patients (n = 299) had elevated DPP4 levels than those with stable angina patients (n = 83). CAD patients even without DM had increased plasma DPP4 activities as compared with nonCAD subjects (10.9 ± 4.9 vs. 6.4 ± 3.1, ng/L, P< 0.01). A linear regression analysis revealed that overall, the DPP4 levels were positively associated with LCL-C and hs-CRP levels as well as syntax scores. A multiple logistic regression analysis demonstrated that plasma DPP4 activity was independent predictor of CAD (odds ratio, 1.56; 95% CI, 1.19–1.73; P<0.01). Our study shows that increased DPP4 activity levels are associated with the presence of CAD and that the plasma DPP4 level serves as a novel biomarker for CAD even without DM.

Increased Circulating Cathepsin K in Patients with Chronic Heart Failure.

Cysteinyl cathepsin K (CatK) is one of the most potent mammalian collagenases involved in cardiovascular disease. Here, we investigated the clinical predictive value of serum CatK levels in patients with chronic heart failure (CHF). We examined 134 patients with CHF, measuring their serum CatK, troponin I, high-sensitive C-reactive protein, and pre-operative N-terminal pro-brain natriuretic peptide levels. The patients were divided into two groups: the 44 patients who showed a left ventricular (LV) ejection fraction (LVEF) < 40% (the “lowLVEF” group) and the 90 patients showing LVEF values ≥ 40% (the “highLVEF” group). The lowLVEF patients had significantly higher serum CatK levels compared to the highLVEF patients (58.4 ± 12.2 vs. 44.7 ± 16.4, P < 0.001). Overall, a linear regression analysis showed that CatK levels correlated negatively with LVEF (r = −0.4, P < 0.001) and positively with LV end-diastolic dimensions (r = 0.2, P < 0.01), LV end-systolic dimensions (r = 0.3, P < 0.001), and left atrial diameters (r = 0.3, P < 0.01). A multiple logistic regression analysis showed that CatK levels were independent predictors of CHF (odds ratio, 0.90; 95% confidence interval, 0.84–0.95; P < 0.01). These data indicate that elevated levels of CatK are closely associated with the presence of CHF and that the measurement of circulating CatK provides a noninvasive method of documenting and monitoring the extent of cardiac remodeling and dysfunction in patients with CHF.

Combined Effects of Short-term Calorie Restriction and Exercise on Insulin Action in Normal Rats

The present study examined the effect of combination of short-term calorie restriction (CR) and moderate exercise on insulin action in normal rats. Rats were divided randomly into 4 groups: ad libitum, sedentary (A-Sed); calorie restriction, sedentary (CR-Sed); ad libitum, exercise (A-Ex); and calorie restriction, exercise (CR-Ex). Rats in the exercise groups were run on a rodent treadmill. Rats in the CR groups were fed every alternate day. Oral glucose tolerance test (OGTT) showed improvements in both CR-Sed and A-Ex groups compared with the A-Sed group; no further improvement in glucose tolerance was observed in the CR-Ex group. In contrast, glucose infusion rates (GIRs) determined by the hyperinsulinemic-euglycemic clamp method indicated that the GIR of the CR and exercise combination was significantly better than that of the sole intervention of CR or exercise. There was no difference in the levels of fasting glucose, insulin, or high-molecular weight forms of adiponectin among the 4 groups. Protein expression of GLUT-4 in the skeletal muscle increased by exercise, but not by CR. Our findings indicate that the combination of exercise and CR may be effective in enhancing insulin sensitivity at the skeletal muscle in normal subjects.

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

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.

Increased dipeptidyl peptidase-4 accelerates diet-related vascular aging and atherosclerosis in ApoE-deficient mice under chronic stress

OBJECTIVES Exposure to psychosocial stress is a risk factor for cardiovascular disease. Given that dipeptidyl peptidase-4 (DPP4) regulates several intracellular signaling pathways associated with glucagon-like peptide-1 (GLP-1) metabolism, we investigated the role of DPP4 in stress-related vascular senescence and atherosclerosis in apolipoprotein E-deficient (ApoE-/-) mice. METHODS AND RESULTS ApoE-/- mice fed a high-fat (HF) diet were randomly assigned to one of non-stress and immobilized stress groups for 12weeks. Chronic stress accelerated vascular senescence and atherosclerotic plaque growth at the aortic roots. Stressed mice had increased levels of plasma DPP4 and decreased levels of plasma GLP-1 and adiponectin (APN) and adipose APN expression. Stress increased plaque macrophage infiltration, neovessel density, and elastin fragmentation, lessened the plaque collagen content, and increased the levels of toll-like receptor-2 (TLR2), TLR4, C-X-C chemokine receptor-4, cathepsins S and K, osteopontin, peroxisome proliferator-activated receptor-α, p16INK4A, p21, and gp91phox mRNAs and/or proteins. Stressed aortas had also increased matrix metalloproteinase-2 (MMP-2) and MMP-9 activities. DPP4 inhibition with anagliptin reversed stress-related atherosclerotic lesion formation, and this benefit was abrogated by APN blocking. In vitro, the GLP-1 receptor agonist exenatide stimulated APN expression in 3T3-L1 cells. CONCLUSIONS These results indicate that the DPP4 inhibition-mediated benefits are likely attributable, at least in part, to attenuation of plaque inflammation, oxidative stress and proteolysis associated with GLP-1-mediated APN production in ApoE-/- mice under stress. Thus, DPP4 will be a novel therapeutic target for the treatment of stress-related cardiovascular disease.

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.