Yabin Wang

In vivo MR and Fluorescence Dual-modality Imaging of Atherosclerosis Characteristics in Mice Using Profilin-1 Targeted Magnetic Nanoparticles.

Aims: This study aims to explore non-invasive imaging of atherosclerotic plaque through magnetic resonance imaging (MRI) and near-infrared fluorescence (NIRF) by using profilin-1 targeted magnetic iron oxide nanoparticles (PF1-Cy5.5-DMSA-Fe3O4-NPs, denoted as PC-NPs) as multimodality molecular imaging probe in murine model of atherosclerosis. Methods and Results: PC-NPs were constructed by conjugating polyclonal profilin-1 antibody and NHS-Cy5.5 fluorescent dye to the surface of DMSA-Fe3O4-nanoparticles via condensation reaction. Murine atherosclerosis model was induced in apoE-/- mice by high fat and cholesterol diet (HFD) for 16 weeks. The plaque areas in aortic artery were detected with Oil Red O staining. Immunofluorescent staining and Western blot analysis were applied respectively to investigate profilin-1 expression. CCK-8 assay and transwell migration experiment were performed to detect vascular smooth muscle cells (VSMCs) proliferation. In vivo MRI and NIRF imaging of atherosclerotic plaque were carried out before and 36 h after intravenous injection of PC-NPs. Oil Red O staining showed that the plaque area was significantly increased in HFD group (p<0.05). Immunofluorescence staining revealed that profilin-1 protein was highly abundant within plaque in HFD group and co-localized with α-smooth muscle actin. Profilin-1 siRNA intervention could inhibit VSMCs proliferation and migration elicited by ox-LDL (p<0.05). In vivo MRI and NIRF imaging revealed that PC-NPs accumulated in atherosclerotic plaque of carotid artery. There was a good correlation between the signals of MRI and ex vivo fluorescence intensities of NIRF imaging in animals with PC-NPs injection. Conclusion: PC-NPs is a promising dual modality imaging probe, which may improve molecular diagnosis of plaque characteristics and evaluation of pharmaceutical interventions for atherosclerosis.

Protective Effects of Low-Frequency Magnetic Fields on Cardiomyocytes from Ischemia Reperfusion Injury via ROS and NO/ONOO−

Background. Cardiac ischemia reperfusion (I/R) injury is associated with overproduction of reactive oxygen species (ROS). Low frequency pulse magnetic fields (LFMFs) have been reported to decrease ROS generation in endothelial cells. Whether LFMFs could assert protective effects on myocardial from I/R injury via ROS regulation remains unclear. Methods. To simulate in vivo cardiac I/R injury, neonatal rat cardiomyocytes were subjected to hypoxia reoxygenation (H/R) with or without exposure to LFMFs. Cell viability, apoptosis index, ROS generation (including O2 − and ONOO−), and NO production were measured in control, H/R, and H/R + LFMF groups, respectively. Results. H/R injury resulted in cardiomyocytes apoptosis and decreased cell viability, whereas exposure to LFMFs before or after H/R injury significantly inhibited apoptosis and improved cell viability (P < 0.05). LFMFs treatment could suppress ROS (including O2 − and ONOO−) generation induced by H/R injury, combined with decreased NADPH oxidase activity. In addition, LFMFs elevated NO production and enhanced NO/ONOO− balance in cardiomyocytes, and this protective effect was via the phosphorylation of endothelial nitric oxide synthase (eNOS). Conclusion. LFMFs could protect myocardium against I/R injury via regulating ROS generation and NO/ONOO− balance. LFMFs treatment might serve as a promising strategy for cardiac I/R injury.

Molecular Imaging of Vulnerable Atherosclerotic Plaques in Vivo with Osteopontin-Specific Upconversion Nanoprobes

Owing to the high mortality rate of cardiovascular diseases, developing novel noninvasive diagnostic methods becomes urgent and mandatory. It is well-known that the rupture of vulnerable plaques directly leads to deadly consequences. However, differentiating vulnerable plaques from stable plaques remains challenging in the clinic. In the current study, osteopontin (OPN), a secreted biomarker associated with macrophages and foamy macrophages, was selected as a target for identifying the vulnerable plaques. A dual modality imaging probe was constructed by covalently attaching an OPN antibody to NaGdF4:Yb,Er@NaGdF4 upconversion nanoparticles. Upon intravenous injection of the resulting probes, upconversion optical imaging was performed to visualize the plaques induced by altering the shear stress in carotid arteries of a mouse model. The imaging studies revealed that the signals of vulnerable and stable plagues induced by lowered shear stress and oscillatory shear stress, respectively, presented significantly different signal intensities, implying that the current probe and imaging strategy are potentially useful for a precise diagnosis of atherosclerosis plaques.

Myocardial Protective Effect of Extracellular Superoxide Dismutase Gene Modified Bone Marrow Mesenchymal Stromal Cells on Infarcted Mice Hearts

Aim: Extracellular superoxide dismutase (ecSOD) is a unique scavenger of superoxide anions and a promising target of gene therapy for ischemia/reperfusion injury (I/R). However, conventional gene therapies have limitation in effectiveness and efficiency. This study aimed to investigate the protective effects of ecSOD gene modified bone marrow mesenchymal stromal cells (BMSCs) on cardiac function improvement in mice infarcted heart. METHODS & RESULTS: BMSCs were isolated from Fluc+ transgenic mice (Tg FVB[Fluc+]) and transfected by adenovirus combined with human ecSOD gene. ELISA was performed to determine ecSOD protein level. Female syngeneic FVB mice were randomized into 5 groups: (1) Sham group (sham); (2) MI group (MI); (3) MI+BMSCs group (BMSC); (4) MI+BMSCs-vector group (BMSC-vector); (5) MI+ BMSCs-ecSOD group (BMSC-ecSOD). MI was accomplished by ligation of the left anterior descending artery. BMSCs (2x106) were injected into the border zone of infarction. In vivo bioluminescence imaging (BLI) was performed to monitor transplanted BMSCs viability. Echocardiography and histological staining revealed that BMSCs-ecSOD significantly reduced myocardial infarction size and improved cardiac function. Lucigenin chemiluminescence, DHE and TUNEL staining demonstrated that BMSCs-ecSOD delivery reduced ROS level and cell apoptosis both in vivo and in vitro. Western blot assay revealed that ecSOD supplementation increased FoxO3a phosphorylation in cardiomyocytes. Moreover, quantitative real-time PCR showed that pro-apoptotic factors (bim and bax) were decreased while the anti-apoptotic factor mir-21 expression was increased after ecSOD intervention. CONCLUSION: Intra-myocardial transplantation of adenovirus-ecSOD transfected BMSCs could exert potential cardiac protection against MI, which may be partly through reduction of oxidative stress and improvement of BMSCs survival.

MRI/optical dual-modality imaging of vulnerable atherosclerotic plaque with an osteopontin-targeted probe based on Fe3O4 nanoparticles

Rupture of vulnerable atherosclerotic plaque is the major pathological cause of luminal thrombosis in acute coronary syndromes. Since foamy macrophages have been identified as a prominent component in vulnerable atherosclerotic lesions and osteopontin (OPN) is reported to be highly expressed in foamy macrophages, OPN could be a potential target for vulnerable atherosclerotic plaque imaging. The current study designed an OPN-specific MRI/optical dual-modality probe to detect vulnerable plaques. Fluorescence imaging revealed that 24xa0h after injection of the Cy5.5-OPN-DMSA-MNPs (COD-MNPs), the atherosclerotic plaques in carotid artery exhibited significant higher signals in high fat diet (HFD) fed mice in comparison to the group injected with Cy5.5-IgG-DMSA-MNPs (CID-MNPs) or normal diet fed group injected with COD-MNPs (1.87xa0±xa00.19xa0×xa01010 vs. 0.74xa0±xa00.04xa0×xa01010, 0.73xa0±xa00.03xa0×xa01010 p/sec/cm2/sr, Pxa0<xa00.05). Meanwhile, MRI displayed stronger T2 contrast enhancement 24xa0h post-injection at the area of atherosclerotic plaques in the carotid of HFD fed group injected with COD-MNPs than group injected with CID-MNPs or normal diet fed group injected with COD-MNPs (post/pre signal ratio: 0.64xa0±xa00.04 vs. 0.95xa0±xa00.02, 0.98xa0±xa00.01, Pxa0<xa00.05). As a dual-modality molecular probe, the resulting COD-MNPs conjugates exhibit promising potentials for noninvasive detection of vulnerable atherosclerotic plaque inxa0vivo.

SIRT1 Activation by Resveratrol Alleviates Cardiac Dysfunction via Mitochondrial Regulation in Diabetic Cardiomyopathy Mice

Background Diabetic cardiomyopathy (DCM) is a major threat for diabetic patients. Silent information regulator 1 (SIRT1) has a regulatory effect on mitochondrial dynamics, which is associated with DCM pathological changes. Our study aims to investigate whether resveratrol, a SRIT1 activator, could exert a protective effect against DCM. Methods and Results Cardiac-specific SIRT1 knockout (SIRT1KO) mice were generated using Cre-loxP system. SIRT1KO mice displayed symptoms of DCM, including cardiac hypertrophy and dysfunction, insulin resistance, and abnormal glucose metabolism. DCM and SIRT1KO hearts showed impaired mitochondrial biogenesis and function, while SIRT1 activation by resveratrol reversed this in DCM mice. High glucose caused increased apoptosis, impaired mitochondrial biogenesis, and function in cardiomyocytes, which was alleviated by resveratrol. SIRT1 deletion by both SIRT1KO and shRNA abolished the beneficial effects of resveratrol. Furthermore, the function of SIRT1 is mediated via the deacetylation effect on peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), thus inducing increased expression of nuclear respiratory factor 1 (NRF-1), NRF-2, estrogen-related receptor-α (ERR-α), and mitochondrial transcription factor A (TFAM). Conclusions Cardiac deletion of SIRT1 caused phenotypes resembling DCM. Activation of SIRT1 by resveratrol ameliorated cardiac injuries in DCM through PGC-1α-mediated mitochondrial regulation. Collectively, SIRT1 may serve as a potential therapeutic target for DCM.

Therapeutic efficacy of apelin on transplanted mesenchymal stem cells in hindlimb ischemic mice via regulation of autophagy

Mesenchymal stem cells (MSCs)-based therapy provides a promising avenue for the management of peripheral arterial disease (PAD). However, engrafted MSCs are subjected to acute cell death in the ischemic microenvironment. Apelin has been shown to protect bone marrow MSCs against apoptosis although the mechanism of action remains elusive. Here we demonstrated that apelin promoted functional survival of AD-MSCs in ischemic hindlimbs and provoked a synergetic effect with AD-MSCs to restore hindlimb blood perfusion and limb functions. Further in vitro studies revealed that a biphasic response in autophagy was induced by apelin in AD-MSCs during hypoxia and hypoxia/reoxygenation (H/R) stages to exert cytoprotective effects against H/R injury. Mechanistically, apelin increased the viability of AD-MSCs via promoting protective autophagy during hypoxia, which was accompanied with activation of AMPK and inhibition of mammalian target of rapamycin (mTOR). To the contrary, apelin suppressed autophagic cell death during reoxygenation, which was accompanied with activation of Akt and inhibition of Beclin1. Our findings indicated that apelin facilitated AD-MSCs-based therapy in PAD, possibly through promoting survival of AD-MSCs by way of autophagy regulation. Our data support the promises of apelin as a novel strategy to improve MSC-based therapy for PAD, possibly through autophagy modulation in MSCs.

Endothelial deletion of mTORC1 protects against hindlimb ischemia in diabetic mice via activation of autophagy, attenuation of oxidative stress and alleviation of inflammation

Abstract Peripheral arterial disease (PAD) complicated with diabetes mellitus (DM) still remains a thorny issue due to lack of effective strategies. Our previous study has demonstrated that inhibition of mTORC1 protected adipose‐derived stromal cells from hindlimb ischemic injury in PAD mice. However, whether inhibition of mTORC1 could protect against PAD in diabetes mellitus and the underlying mechanisms remained elusive. In this study, we employed endothelial‐specific raptor (an essential component of the mTORC1 signaling complex) knockout (KO) mice (Tie2‐mTORC1ko) to investigate whether and how mTORC1 downregulation could alleviate hindlimb ischemic injury in diabetic mice. Tie2‐mTORC1ko mice and their wild‐type littermates were intraperitoneally injected with streptozocin to induce type 1 diabetic model, after which the hyperglycemic mice were randomly allocated to sham operation or PAD operation (femoral artery ligation). The restoration of hindlimb blood perfusion and recovery of limb functions were improved in diabetic Tie2‐mTORC1ko PAD mice with significant improvements of autophagy, angiogenesis and vascular integrity as well as attenuation of apoptosis, inflammation and oxidative stress. In vitro, high glucose combining with hypoxia/serum deprivation treatment (HG+H/SD) significantly triggered apoptosis, reactive oxygen species generation and inflammation while inhibited autophagy and tube formation in HUVECs. The effect could be accentuated and attenuated by mTORC1 over‐expression (TSC2 siRNA) and mTORC1 silencing (raptor siRNA), respectively. Moreover, autophagy inhibitor 3‐MA could simulate the effects of TSC2 siRNA while autophagy inducer rapamycin could mimic the effects of raptor siRNA, suggesting that the beneficial effects of mTORC1 deletion were associated with autophagy induction. In conclusion, our present study demonstrates that endothelial mTORC1 deletion protects against hindlimb ischemic injury in diabetic mice possibly via activation of autophagy, attenuation of oxidative stress and alleviation of inflammation. Therapeutics targeting mTORC1 may therefore represents a promising strategy to rescue limb ischemia in diabetes mellitus. Graphical abstract Figure. No Caption available. HighlightsEndothelial deletion of mTORC1 protects agaisnst limb ischemia in diabetic mice.mTORC1 silencing preserved endothelial viability and function.mTORC1 silencing restored impaired autophagy to scavenge damaged mitochondria.mTORC1 deletion attenuated oxidative stress in ischemsic gastrocnemius muscle.mTORC1‐p70S6K axis exhibited pro‐inflammatory potential.

Activation of Cannabinoid Receptor Type II by AM1241 Ameliorates Myocardial Fibrosis via Nrf2-Mediated Inhibition of TGF-β1/Smad3 Pathway in Myocardial Infarction Mice.

Aims: Myocardial interstitial fibrosis is a major histologic landmark resulting in cardiac dysfunction after myocardial infarction (MI). Activation of cannabinoid receptor type II (CB2 receptor) have been demonstrated to reduce fibrosis in hepatic cirrhotic rat. However, the anti-fibrotic effect of CB2 receptor activation in infarcted hearts was still unclear. In this study, we aimed to investigate the effects of a CB2 receptor selective agonist AM1241 on myocardial fibrosis post MI in mice. Methods: Echocardiograph was conducted to assess cardiac function. Fibrosis markers such as type I and type III collagen, fibronectin, Plasminogen activator inhibitor(PAI)-1 and tissue inhibitor of metalloprotease(TIMP)-1 were examined by Western blot, while collagens were directly observed by Sirius-red staining. Primary cultured cardiac fibroblasts(CFs) were subjected to hypoxia/serum deprivation (H/SD) injury to simulate ischemic conditions in vivo. Nrf2 siRNA were applied to explore the role of Nrf2 and TGF-β1/Smad3 pathway in this process. Results: Echocardiography showed that AM1241 significantly improved cardiac function, suppressed the expression of fibrosis markers such as collagen I and collagen III, fibronectin, PAI-1 and TIMP-1 in mice with MI. In cardiac fibroblasts subjected to H/SD injury, AM1241 reduced the elevated levels of α-SMA, collagen I and collagen III, which were partially abrogated by the Nrf2 siRNA transfection. Furthermore, AM1241 not only activated and accelerated the translocation of Nrf2 to nucleus, but also inhibited TGF-β1/ Smad3 pathway in an Nrf2 dependent manner. Conclusion: CB2 receptor agonist AM1241 alleviated myocardial interstitial fibrosis via Nrf2 -mediated down-regulation of TGF-β1/Smad3 pathway, which suggested that CB2 receptor activation might represent a promising target for retarding cardiac fibrosis after MI.

Activation of aldehyde dehydrogenase 2 slows down the progression of atherosclerosis via attenuation of ER stress and apoptosis in smooth muscle cells

Aldehyde dehydrogenase 2 (ALDH2) is a key mitochondrial enzyme in the metabolism of aldehydes and may have beneficial cardiovascular effects for conditions such as cardiac hypertrophy, heart failure, myocardial I/R injury, reperfusion, arrhythmia, coronary heart disease and atherosclerosis. In this study we investigated the role of ALDH2 in the progression of atherosclerosis and the underlying mechanisms, with a focus on endoplasmic reticulum (ER) stress. A clinical study was performed in 248 patients with coronary heart disease. The patients were divided into two groups according to their ALDH2 genotype. Baseline clinical characteristics and coronary angiography were recorded, and the coronary artery Gensini score was calculated. Serum levels of 4-hydroxy-2-nonenal (4-HNE) were detected. The clinical study revealed that the mutant ALDH2 genotype was an independent risk factor for coronary heart disease. ALDH2 gene polymorphism is closely associated with atherosclerosis and the severity of coronary artery stenosis. Serum levels of 4-HNE were significantly higher in patients with the mutant ALDH2 genotype than in patients with the wild-type ALDH2 genotype. As an in vitro model of atherosclerosis, rat smooth muscle cells (SMCs) were treated with oxygenized low-density lipoprotein (ox-LDL), which significantly elevated the levels of ER markers glucose-regulated protein78 (GRP78), protein kinase R-like ER kinase (PERK), phosphorylated eukaryotic translation initiation factor α subunit (p-eIF2α), activating transcription factor-4 (ATF-4), CEBP homologous protein (CHOP) and 4-HNE in the cells. All the ox-LDL-induced responses were significantly attenuated in the presence of Alda-1 (an ALDH2 activating agent), and accentuated in the presence of daidzin (an ALDH2 inhibitor). Furthermore, pretreatment with ALDH2 activator Alda-1 significantly decreased ox-LDL-induced apoptosis. Similarly, overexpression of ALDH2 protected SMCs against ox-LDL-induced ER stress as well as ER stress-induced apoptosis. These findings suggest that ALDH2 may slow the progression of atherosclerosis via the attenuation of ER stress and apoptosis in smooth muscle cells.