Loss of myeloid Tsc2 predisposes to angiotensin II-induced aortic aneurysm formation in mice.

RATIONALE: Genetic studies have proved the involvement of Tuberous sclerosis complex subunit 2 (Tsc2) in aortic aneurysm. However, the exact role of macrophage Tsc2 in the vascular system remains unclear. Here, we examined the potential function of macrophage Tsc2 in the development of aortic remodeling and aortic aneurysms. METHODS AND RESULTS: Conditional gene knockout strategy combined with histology and whole-transcriptomic analysis showed that Tsc2 deficiency in macrophages aggravated the progression of aortic aneurysms along with an upregulation of proinflammatory cytokines and matrix metallopeptidase-9 in the angiotensin II-induced mouse model. G protein-coupled receptor 68 (Gpr68), a proton-sensing receptor for detecting the extracellular acidic pH, was identified as the most up-regulated gene in Tsc2 deficient macrophages compared with control macrophages. Additionally, Tsc2 deficient macrophages displayed higher glycolysis and glycolytic inhibitor 2-deoxy-D-glucose treatment partially attenuated the level of Gpr68. We further demonstrated an Tsc2-Gpr68-CREB network in macrophages that regulates the inflammatory response, proteolytic degradation and vascular homeostasis. Gpr68 inhibition largely abrogated the progression of aortic aneurysms caused by Tsc2 deficiency in macrophages. CONCLUSIONS: The findings reveal that Tsc2 deficiency in macrophages contributes to aortic aneurysm formation, at least in part, by upregulating Gpr68 expression, which subsequently drives proinflammatory processes and matrix metallopeptidase activation. The data also provide a novel therapeutic strategy to limit the progression of the aneurysm resulting from Tsc2 mutations.

Correction: Loss of PARP-1 attenuates diabetic arteriosclerotic calcification via Stat1/Runx2 axis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Loss of PARP-1 attenuates diabetic arteriosclerotic calcification via Stat1/Runx2 axis.

Accelerated atherosclerotic calcification is responsible for plaque burden, especially in diabetes. The regulatory mechanism for atherosclerotic calcification in diabetes is poorly characterized. Here we show that deletion of PARP-1, a main enzyme in diverse metabolic complications, attenuates diabetic atherosclerotic calcification and decreases vessel stiffening in mice through Runx2 suppression. Specifically, PARP-1 deficiency reduces diabetic arteriosclerotic calcification by regulating Stat1-mediated synthetic phenotype switching of vascular smooth muscle cells and macrophage polarization. Meanwhile, both vascular smooth muscle cells and macrophages manifested osteogenic differentiation in osteogenic media, which was attenuated by PARP-1/Stat1 inhibition. Notably, Stat1 acts as a positive transcription factor by directly binding to the promoter of Runx2 and promoting atherosclerotic calcification in diabetes. Our results identify a new function of PARP-1, in which metabolism disturbance-related stimuli activate the Runx2 expression mediated by Stat1 transcription to facilitate diabetic arteriosclerotic calcification. PARP-1 inhibition may therefore represent a useful therapy for this challenging complication.

PARP-1 (Poly[ADP-Ribose] Polymerase 1) Inhibition Protects From Ang II (Angiotensin II)-Induced Abdominal Aortic Aneurysm in Mice.

Abdominal aortic aneurysm (AAA) is a common vascular degenerative disease. PARP-1 (poly[ADP-ribose] polymerase 1) is a nuclear enzyme, which plays a critical role in vascular diseases. We hypothesized that PARP-1 inhibition might have protective effects on AAA. In vivo, Ang II (angiotensin II) was continuously infused by a micropump for 28 days to induce AAA in mice. In vitro, aortic endothelial cells and smooth muscle cells were stimulated by Ang II for 24 hours. Ang II infusion increased PARP-1 expression and activity and successfully induced AAA formation partly with a hemorrhage in ApoE-/- mice. Genetic deletion of PARP-1 markedly reduced the AAA incidence, abdominal aortic diameter, macrophage infiltration, ICAM-1 (intercellular adhesion molecule 1) and VCAM-1 (vascular adhesion molecule 1) expression, and MMP (matrix metalloproteinase) expression, as well as MMP activity; but increased smooth muscle cells content and collagens expression in AAA. PARP-1 inhibition by PJ-34 also exerted a protective effect on AAA in mice. In aortic endothelial cells, Ang II-induced oxidative stress and DNA damage, resulting in increased PARP-1 expression and activity. Compared with the control, Ang II increased TNF-α (tumor necrosis factor α) and IL-6 (interleukin-6) secretions, ICAM-1 expression and THP-1 (human acute monocytic leukemia cell line) cells adhesion, while PARP-1 inhibition by siRNA reduced the inflammatory response probably through inhibition of the phosphorylation of ERK (extracellular signal-regulated kinase), NF-κB (nuclear factor-κB), and Akt signaling pathways. In smooth muscle cells, Ang II promoted cell migration, proliferation, and apoptosis, reduced collagens expression, but increased MMPs expression, while PARP-1 deletion alleviated these effects partly by reducing NF-κB-targeted MMP-9 expression. PARP-1 inhibition might be a feasible strategy for the treatment of AAA.

Peptidyl-prolyl isomerase Pin1 deficiency attenuates angiotensin II-induced abdominal aortic aneurysm formation in ApoE-/- mice.

Peptidyl-prolyl isomerase Pin1 has been reported to be associated with endothelial dysfunction. However, the role of smooth muscle Pin1 in the vascular system remains unclear. Here, we examined the potential function of Pin1 in smooth muscle cells (SMCs) and its contribution to abdominal aortic aneurysm (AAA) pathogenesis. The level of Pin1 expression was found to be elevated in human AAA tissues and mainly localized to SMCs. We constructed smooth muscle-specific Pin1 knockout mice to explore the role of this protein in AAA formation and to elucidate the underlying mechanisms. AAA formation and elastin degradation were hindered by Pin1 depletion in the angiotensin II-induced mouse model. Pin1 depletion reversed the angiotensin II-induced pro-inflammatory and synthetic SMC phenotype switching via the nuclear factor (NF)-κB p65/Klf4 axis. Moreover, Pin1 depletion inhibited the angiotensin II-induced matrix metalloprotease activities. Mechanically, Pin1 deficiency destabilized NF-κB p65 by promoting its polyubiquitylation. Further, we found STAT1/3 bound to the Pin1 promoter, revealing that activation of STAT1/3 was responsible for the increased expression of Pin1 under angiotensin II stimulation. Thus, these results suggest that Pin1 regulates pro-inflammatory and synthetic SMC phenotype switching and could be a novel therapeutic target to limit AAA pathogenesis.

Irisin inhibits high glucose-induced endothelial-to-mesenchymal transition and exerts a dose-dependent bidirectional effect on diabetic cardiomyopathy.

Emerging evidence indicates that irisin provides beneficial effects in diabetes. However, whether irisin influences the development of diabetic cardiomyopathy (DCM) remains unclear. Therefore, we investigated the potential role and mechanism of action of irisin in diabetes-induced myocardial dysfunction in mice. Type 1 diabetes was induced in mice by injecting streptozotocin, and the diabetic mice were administered recombinant r-irisin (low or high dose: 0.5 or 1.5 µg/g body weight/day, I.P.) or PBS for 16 weeks. Irisin treatment did not alter blood glucose levels in the diabetic mice. However, the results of echocardiographical and histopathological assays indicated that low-dose irisin treatment alleviated cardiac fibrosis and left ventricular function in the diabetic mice, whereas high-dose irisin failed to mitigate the ventricular function impairment and increased collagen deposition. The potential mechanism underlying the effect of low-dose irisin involved irisin-mediated inhibition of high glucose-induced endothelial-to-mesenchymal transition (EndMT); conversely, high-dose irisin treatment enhanced high glucose-induced MMP expression by stimulating MAPK (p38 and ERK) signalling and cardiac fibroblast proliferation and migration. Low-dose irisin alleviated DCM development by inhibiting high glucose-induced EndMT. By contrast, high-dose irisin disrupted normal MMP expression and induced cardiac fibroblast proliferation and migration, which results in excess collagen deposition. Thus, irisin can inhibit high glucose-induced EndMT and exert a dose-dependent bidirectional effect on DCM.

Paeonol exerts potential activities to inhibit the growth, migration and invasion of human gastric cancer BGC823 cells via downregulating MMP­2 and MMP­9.

Paeonol (Pae) is an herbal extract that has attracted extensive attention for its anti­cancer effects demonstrated by a number of studies, which have predominantly demonstrated inhibition of cell proliferation and induction of apoptosis. The influence of Pae on cancer cell metastasis has been less widely reported. The present study aimed to investigate the under­reported effects of Pae on the growth, invasion and migration of poorly differentiated BGC823 gastric cancer cells with strong invasive and metastatic abilities. The anti­proliferative and pro­apoptotic effects of Pae on BGC823 cells were verified by Cell Counting kit­8 and Annexin V­fluorescein isothiocyanate/propidium iodide assays. Cell scratch­wound healing and Transwell methods were applied, and it was demonstrated that Pae could exert inhibitory activities on the invasion and migration of BGC823 cells. Furthermore, it was indicated by western blot analysis that Pae could downregulate the protein expression levels of matrix metalloproteinase (MMP)­2 and ­9 in a concentration­dependent manner, which may support a novel potential mechanism accounting for its anti­cancer effects on gastric cancer.

Allicin improves carotid artery intima-media thickness in coronary artery disease patients with hyperhomocysteinemia.

Homocysteine (Hcy) is an important and independent risk factor for atherosclerotic diseases, such as coronary artery disease and ischemic cerebrovascular disease. Increased carotid artery intima-media thickness (IMT) is a non-invasive marker of systemic atherosclerosis. Allicin treatment may decrease serum Hcy levels and improve impaired endothelial function in rats with hyperhomocysteinemia (HHcy). The present study hypothesized that allicin has an anti-atherosclerotic effect in coronary heart disease and tested the effects of allicin treatment on carotid artery IMT and plasma Hcy levels in coronary heart disease patients with HHcy. Sixty-two coronary heart disease patients with HHcy were randomly divided into an allicin group and a control group. All patients underwent diagnostic assessment, plasma Hcy assay, blood lipid measurement and B-mode ultrasound of the carotid artery prior to and after treatment. Plasma Hcy levels were determined by high-performance liquid chromatography and fluorescence detection. Carotid artery IMT was calculated using an automated algorithm based on a validated edge-detection technique. After 12 weeks, significant decreases in carotid artery IMT, plasma Hcy levels, total cholesterol and triglycerides were observed in the allicin group (all P<0.05), and the decreases in the allicin group were significantly greater than those in the control group (all P<0.01). These findings suggested that reducing plasma Hcy levels may be useful for preventing the generation and development of atherosclerosis in patients with coronary heart disease. Allicin was able to decrease Hcy levels, total cholesterol and triglycerides as well as carotid artery IMT.

Poly(ADP-ribose) polymerase 1 deficiency increases nitric oxide production and attenuates aortic atherogenesis through downregulation of arginase II.

Poly (ADP-ribose) polymerase (PARP) plays an important role in endothelial dysfunction, leading to atherogenesis and vascular-related diseases. However, whether PARP regulates nitric oxide (NO), a key regulator of endothelial function, is unclear so far. We investigated whether inhibition of PARP-1, the most abundant PARP isoform, prevents atherogenesis by regulating NO production and tried to elucidate the possible mechanisms involved in this phenomenon. In apolipoprotein E-deficient (apoE-/- ) mice fed a high-cholesterol diet for 12 weeks, PARP-1 inhibition via treatment with 3,4-dihydro-54-(1-piperindinyl) butoxy-1(2H)-isoquinoline (DPQ) or PARP-1 gene knockout reduced aortic atherosclerotic plaque areas (49% and 46%, respectively). Both the groups showed restored NO production in mouse aortas with reduced arginase II (Arg II) expression compared to that in the controls. In mouse peritoneal macrophages and aortic endothelial cells (MAECs), PARP-1 knockout resulted in lowered Arg II expression. Moreover, phosphorylation of endothelial NO synthase (eNOS) was preserved in the aortas and MAECs when PARP-1 was inhibited. Reduced NO production in vitro due to PARP-1 deficiency could be restored by treating the MAECs with oxidized low-density lipoprotein treatment, but this effect could not be achieved with peritoneal macrophages, which was likely due to a reduction in the expression of induced NOS expression. Our findings indicate that PARP-1 inhibition may attenuate atherogenesis by restoring NO production in endothelial cells and thus by reducing Arg II expression and consequently arginase the activity.

Inhibition of myocyte-specific enhancer factor 2A improved diabetic cardiac fibrosis partially by regulating endothelial-to-mesenchymal transition.

Cardiac fibrosis is an important pathological process of diabetic cardiomyopathy, the underlying mechanism remains elusive. This study sought to identify whether inhibition of Myocyte enhancer factor 2A (MEF2A) alleviates cardiac fibrosis by partially regulating Endothelial-to-mesenchymal transition (EndMT). We induced type 1 diabetes mellitus using the toxin streptozotocin (STZ) in mice and injected with lentivirus-mediated short-hairpin RNA (shRNA) in myocardium to inhibit MEF2A expression. Protein expression, histological and functional parameters were examined twenty-one weeks post-STZ injection. We found that Diabetes mellitus increased cardiac MEF2A expression, aggravated cardiac dysfunction and myocardial fibrosis through the accumulation of fibroblasts via EndMT. All of these features were abolished by MEF2A inhibition. MEF2A gene silencing by shRNA in cultured human umbilical vein endothelial cells (HUVECs) ameliorated high glucose-induced phenotypic transition and acquisition of mesenchymal markers through interaction with p38MAPK and Smad2. We conclude that inhibition of endothelial cell-derived MEF2A might be beneficial in the prevention of diabetes mellitus-induced cardiac fibrosis by partially inhibiting EndMT through interaction with p38MAPK and Smad2.

Prohibitin overexpression improves myocardial function in diabetic cardiomyopathy.

Prohibitin (PHB) is a highly conserved protein implicated in various cellular functions including proliferation, apoptosis, tumor suppression, transcription, and mitochondrial protein folding. However, its function in diabetic cardiomyopathy (DCM) is still unclear. In vivo, type 2 diabetic rat model was induced by using a high-fat diet and low-dose streptozotocin. Overexpression of the PHB protein in the model rats was achieved by injecting lentivirus carrying PHB cDNA via the jugular vein. Characteristics of type 2 DCM were evaluated by metabolic tests, echocardiography and histopathology. Rats with DCM showed severe insulin resistance, left ventricular dysfunction, fibrosis and apoptosis. PHB overexpression ameliorated the disease. Cardiofibroblasts (CFs) and H9c2 cardiomyoblasts were used in vitro to investigate the mechanism of PHB in altered function. In CFs treated with HG, PHB overexpression decreased expression of collagen, matrix metalloproteinase activity, and proliferation. In H9c2 cardiomyoblasts, PHB overexpression inhibited apoptosis induced by HG. Furthermore, the increased phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 was significantly decreased and the inhibited phosphorylation of Akt was restored in DCM. Therefore, PHB may be a new therapeutic target for human DCM.

Protective effect of allicin on high glucose/hypoxia-induced aortic endothelial cells via reduction of oxidative stress.

This study was designed to explore the protective effect of allicin on aortic endothelial cell injury induced by high glucose/hypoxia and to investigate the corresponding mechanisms. The primary-cultured murine aortic endothelial cells were subcultured. The third passage of cells was adopted and randomly divided into five groups: The normal group (NG), the mannitol group (MG), the high-glucose/hypoxia group (HG), the allicin group (AG) and the protein kinase C (PKC) inhibitor group (GG). The general morphology was observed under an inverted phase-contrast microscope and cell viability was assessed using the MTT assay. Intracellular reactive oxygen species (ROS) levels in the endothelial cells were quantified using dihydroethidium staining. The levels of 8-hydroxydeoxyguanosine (8-OHdG), nuclear factor-κB (NF-κB), NADPH oxidase 4 (Nox4) and hypoxia-inducible factor-1α (HIF-1α) and the activity of PKC were measured using ELISA. A quantitative polymerase chain reaction (qPCR) was adopted to evaluate the mRNA expression of Nox4, HIF-1α and NF-κB. The altered cell morphology observed in HG was notably ameliorated in the AG and GG. The protein levels of 8-OHdG, NF-κB, Nox4, HIF-1α and PKC in the HG were higher than those in the other groups. Furthermore, the cell viability in the AG was significantly increased and the protein levels of 8-OHdG, NF-κB, Nox4, HIF-1α and PKC were significantly decreased compared with those in the HG. The ROS production was found to be increased in the HG cells, while there was a significant decrease in the AG cells. These data indicate that allicin exerts a protective effect against high glucose/hypoxia-induced injury in aortic endothelial cells through its antioxidative action, which may involve the inhibition of the PKC pathway and regulation of HIF-1α.

Effects of allicin on hyperhomocysteinemia-induced experimental vascular endothelial dysfunction.

This study was designed to investigate the effect and mechanism of allicin on hyperhomocysteinemia-induced experimental vascular endothelial dysfunction in rats. Fifty male Wistar rats were randomly divided into five groups: the normal control rats (NC), the high-methionine-diet rats (Met), the high-methionine-diet rats treated with folic acid, vitaminB6 and vitaminB12 (Met+F), or with low-dose allicin (Met+L), or with high-dose allicin (Met+H). After 6 weeks, we collected blood samples of all groups to determine plasma endothelin (ET), serum homocysteine (Hcy), nitric oxide (NO), superoxide dismutase (SOD), malondialdehyde (MDA), and detected the expression of basic fibroblast growth factors (bFGF), transforming growth factor beta (TGF-ß), tumor necrosis factor-alpha (TNF-α), and intercellular adhesion molecule-1 (ICAM-1) in the aorta. The Hcy and the expression of TGF-ß in both the Met+L and Met+H groups were significantly lower than the Met and Met+F groups. The ET, ET/NO ratio and the MDA levels of the Met+L and Met+H groups were significantly lower than the Met group. The SOD and NO levels and the expression of bFGF, TNF-α and ICAM-1 of the Met+L and Met+H groups were significantly higher than the Met group. Our data indicate that allicin inhibits lipid peroxidation induced by hyperhomocysteinemia and regulates the excretion and equilibrium of ET and NO, and suggest that allicin might be useful in the prevention of endothelial dysfunction caused by hyperhomocysteinemia.

Neuroprotective effects of the Chinese Yi-Qi-Bu-Shen recipe extract on injury of rat hippocampal neurons induced by hypoxia/reoxygenation.

OBJECTIVE: To explore the protective effects of the Chinese Yi-Qi-Bu-Shen recipe (YB) against neuronal injury induced by hypoxia-reoxygenation, which has shown beneficial effect in improving the brain function of type 2 diabetics likely through its antihyperglycemic, antioxidant activity, and investigate its mechanisms. METHODS: The bilateral hippocampus was collected from newborn rats to establish single cell suspension. On the 10th day, the primarily cultured hippocampal neurons were randomly divided into five groups: the normal group (NG), the hypoxia/reoxygenation group (HG), and groups protected with small, medium and large dosages of YB (SG, MG and LG, respectively). The YB-protected groups were treated with different concentrations of YB containing serum before reoxygenation. The metabolic rate of MTT, the malondialdehyde (MDA) content, and the activity of superoxide dismutase (SOD) and lactate dehydrogenase (LDH) were measured with assay kits. The apoptosis rate of hippocampal neurons were tested using flow cytometry analysis. RT-PCR was used to evaluate the mRNA expressions of bcl-2 and bax genes. RESULTS: The SOD activity, the cell survival rate, the bcl-2/bax ratio, and the bcl-2mRNA expression in the HG group were significantly lower (all P<0.01), but the levels of MDA and LDH, the apoptosis rate, and the bax mRNA expression were higher (all P<0.01) than those in the NG group. The SOD activity, the cell survival, the bcl-2mRNA expression, and the bcl-2/bax ratio were significantly higher in all of the YB-protected groups (all P<0.01), but the level of MDA and LDH, the apoptosis rate, and the bax mRNA expression were lower (P<0.01, P<0.05) than those in the HG group in a dose dependent manner. CONCLUSION: The YB extract has a protective effect on hippocampal neurons against injury induced by hypoxia/reoxygenation, through its antioxidant activity and the regulation of apoptosis.