Hongxian Wu

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 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.

Therapeutic angiogenesis by autologous adipose-derived regenerative cells: comparison with bone marrow mononuclear cells

Transplantation of adipose-derived regenerative cell (ADRC) enhances ischemia-induced angiogenesis, but the underlying mechanism remains unknown. Here, we compared the efficacy between ADRC and bone marrow mononuclear cell (BM-MNC) transplantation in rabbits model of hindlimb ischemia and examined the possible roles of alternative phenotypic macrophages polarization in ADRC-mediated angiogenesis using mice model of hindlimb ischemia. ADRCs and BM-MNCs were isolated from New Zealand White rabbits and C57BL/6J mice. In rabbit studies, our data showed that ADRCs could incorporate into the endothelial vasculature in vitro and in vivo. Both ADRC-conditioned media (CM) and BM-MNC-CM enhanced the migratory ability and interrupted the process of apoptosis in human umbilical vein endothelial cells. Four weeks after cell transplantation, augmentation of postnatal neovascularization was observed in the ischemic muscle injected with either ADRCs or BM-MNCs. In mice studies, we presented that ADRCs polarized into the IL-10-releasing M2 macrophages through PGE2-EP2/4 axis and suppressed the expressions of TNF-α and IL-6 in the ischemic muscle. Gene expressions of several angiogenic cytokines were amplified in the macrophages cultured in ADRC-CM rather than BM-MNC-CM. Blockade of IL-10 using neutralizing MAb attenuated the ADRC-mediated angiogenesis and caused muscle apoptosis in vivo. In conclusion, ADRC transplantation harvested similar effect of neovascularization augmentation compared with BM-MNC in experimental rabbit model of hindlimb ischemia; ADRC displayed a unique immunoregulatory manner of accelerating IL-10-releasing M2 macrophages polarization through the PGE2-EP2/4 axis.

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.

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.

Xanthine oxidase inhibition by febuxostat attenuates stress-induced hyperuricemia, glucose dysmetabolism, and prothrombotic state in mice

Chronic stress is closely linked to the metabolic syndrome, diabetes, hyperuricemia and thromboembolism, but the mechanisms remain elusive. We reported recently that stress targets visceral adipose tissue (VAT), inducing lipolysis, low-grade inflammation with production of inflammatory adipokines, metabolic derangements such as insulin resistance, and prothrombotic state. In the present study, we hypothesized the involvement of VAT xanthine oxidoreductase (XOR), a source of reactive oxygen species (ROS) and uric acid (UA) in the above processes. Restraint stress in mice resulted in upregulation of XOR and xanthine oxidase activity, accumulation of ROS in VAT as well as liver and intestine, increase in serum UA levels, upregulation of NADPH oxidase subunits and downregulation of antioxidant enzymes. Immunohistochemistry and RT-PCR analysis also showed that restraint stress induced VAT monocyte accumulation and proinflammatory adipokine production, resulting in reduced insulin sensitivity and induction of plasminogen activator inhibitor-1 and tissue factor in VAT. Treatment with febuxostat, a potent XO inhibitor, suppressed stress-induced ROS production and VAT inflammation, resulting in improvement of serum UA levels, insulin sensitivity, and prothrombotic tendency. Our results suggest that stress perturbs glucose and UA metabolism, and promotes prothrombotic status, and that XO inhibition by febuxostat might be a potential therapy for stress-related disorders.

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.

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.

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 Adrβ3 (β3‐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 Adrβ3/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 Adrβ3 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‐Kithigh CD48low CD150high 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 Adrβ3 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 Adrβ3/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.

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.