Hong-Xia Wang

Apelin protects heart against ischemia/reperfusion injury in rat

Apelin, the endogenous ligand of the G protein-coupled APJ receptor, is a peptide mediator with emerging regulatory actions in the heart. We aimed to determine whether the endogenous apelin/APJ system is an intrinsic protective pathway in ischemic/reperfusion injury. A Langendorff model of perfused isolated rat hearts and primary cultured myocardial cells from neonatal rats were used. Cardiac function was monitored and apelin/APJ expression was determined by real-time PCR and Western blot analysis. In rats under I/R, cardiac function was significantly decreased as compared with controls, and APJ was over-expressed at both the mRNA and protein levels (by 7-fold and 35%, respectively, both p<0.01). However, pre-administration of apelin (30pmol/L) greatly ameliorated the reduced heart function. To gain mechanistic insight into the cardio-protective effects of apelin/APJ, cultured cardiomyocytes were treated with apelin (30 pmol/L), and those under hypoxia/re-oxygenation showed H/R-induced apoptosis and up-regulated apelin/APJ mRNA expression by 6-fold and 7-fold, respectively (both p<0.01). And lactate dehydrogenase leakage was greatly increased as well. Meanwhile, apoptosis, the generation of reactive oxygen species and malonaldehyde content as well as lactate dehydrogenase leakage were inhibited by apelin. Furthermore, apelin enhanced superoxide dismutase activity and phosphorylation of extracellular signal-regulated kinase 1/2 and Akt after hypoxia/re-oxygenation. In conclusion, apelin/APJ has protective effects in ischemic heart disease and might constitute an important therapy target.

Inhibition of 12/15 lipoxygenase by baicalein reduces myocardial ischemia/reperfusion injury via modulation of multiple signaling pathways.

Abstract12/15-Lipoxygenase (LOX) is a member of the LOX family that catalyzes the step from arachidonic acid to hydroxy-eicosatetraenoic acids (HETEs). Previous studies demonstrated that 12/15-LOX plays a critical role in the development of atherosclerosis, hypertension, heart failure, and other diseases; however, its role in myocardial ischemic injury was contraversal. Here, we investigated the inhibition of 12/15-LOX by baicalein on acute cardiac injury and dissected its molecular mechanism. In a mouse model of acute ischemia/reperfusion (I/R) injury, 12/15-LOX was significantly upregulated in the peri-infarct area surrounding the primary infarction. In cultured cardiac myocytes, baicalein suppressed apoptosis and caspase 3 activity in response to simulated ischemia/reperfusion (I/R). Moreover, administration of 12/15-LOX inhibitor, baicalein, significantly attenuated myocardial infarct size induced by I/R injury. Moreover, baicalein treatment significantly inhibited cardiomyocyte apoptosis, inflammatory responses and oxidative stress in the heart after I/R injury. The mechanisms underlying these effects were associated with the activation of ERK1/2 and AKT pathways and inhibition of activation of p38 MAPK, JNK1/2, and NF-kB/p65 pathways in the I/R-treated hearts and neonatal cardiomyoctes. Our data indicated that 12/15-LOX inhibitor baicalein can prevent myocardial I/R injury by modulation of multiple mechanisms, and suggest that baicalein could represent a novel therapeutic drug for acute myocardial infarction.

Cardioprotective effects of adipokine apelin on myocardial infarction

Angiogenesis plays an important role in myocardial infarction. Apelin and its natural receptor (angiotensin II receptor-like 1, AGTRL-1 or APLNR) induce sprouting of endothelial cells in an autocrine or paracrine manner. The aim of this study is to investigate whether apelin can improve the cardiac function after myocardial infarction by increasing angiogenesis in infarcted myocardium. Left ventricular end-diastolic pressure (LVEDP), left ventricular end systolic pressure (LVESP), left ventricular developed pressure (LVDP), maximal left ventricular pressure development (±LVdp/dtmax), infarct size, and angiogenesis were evaluated to analyze the cardioprotective effects of apelin on ischemic myocardium. Assays of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 5-bromo-2′-deoxyuridine incorporation, wound healing, transwells, and tube formation were used to detect the effects of apelin on proliferation, migration, and chemotaxis of cardiac microvascular endothelial cells. Fluorescein isothiocyanate-labeled bovine serum albumin penetrating through monolayered cardiac microvascular endothelial cells was measured to evaluate the effects of apelin on permeability of microvascular endothelial cells. In vivo results showed that apelin increased ±LV dp/dtmax and LVESP values, decreased LVEDP values (all p < 0.05), and promoted angiogenesis in rat heart after ligation of the left anterior descending coronary artery. In vitro results showed that apelin dose-dependently enhanced proliferation, migration, chemotaxis, and tube formation, but not permeability of cardiac microvascular endothelial cells. Apelin also increased the expression of vascular endothelial growth factor receptors-2 (VEGFR2) and the endothelium-specific receptor tyrosine kinase (Tie-2) in cardiac microvascular endothelial cells. These results indicated that apelin played a protective role in myocardial infarction through promoting angiogenesis and decreasing permeability of microvascular endothelial cells via upregulating the expression of VEGFR2 and Tie-2 in cardiac microvascular endothelial cells.

CHIP Enhances Angiogenesis and Restores Cardiac Function After Infarction in Transgenic Mice

Background: Carboxyl terminus of Hsp70-interacting protein (CHIP) is a chaperone/ubiquitin ligase that plays an important role in stress-induced apoptosis. However, the effect of CHIP on angiogenesis, cardiac function and survival 4 weeks after myocardial infarction (MI) remain to be explored. Methods: Wild-type (WT) and transgenic mice (TG) with cardiac-specific overexpression of CHIP were used for coronary artery ligation. The cardiac function, cardiomyocyte apoptosis, inflammation and angiogenesis were examined by echocardiography, histological analysis, real-time PCR and Western blot analysis. Results: At 4 weeks of after coronary artery ligation, echocardiography demonstrated that cardiac remodeling and dysfunction were prevented in TG mice compared with WT mice. The infarct size, cardiomyocyte apoptosis and inflammation were significantly reduced in TG mice than in WT mice. The survival rate after MI in TG mice was higher than that of WT mice. Furthermore, the levels of p53 protein was markedly decreased, but the expression of HIF-1α and VEGF, and the formation of capillary and arteriole after MI were significantly enhanced in TG mice compared with WT mice. Conclusion: We report the first in vivo evidence that CHIP enhances angiogenesis, inhibits inflammation, restores cardiac function, and improves survival at 4 weeks after MI. The present study expands on previous results and defines a novel mechanism. Thus, increased CHIP level may provide a novel therapeutic approach for left ventricular dysfunction after MI.

MicroRNA Let-7i Negatively Regulates Cardiac Inflammation and Fibrosis

Angiotensin II stimulates fibroblast proliferation and substantially alters gene expression patterns leading to cardiac remodeling, but the mechanisms for such differences are unknown. MicroRNAs are a novel mechanism for gene expression regulation. Herein, we tested the miRNA and mRNA expression patterns in mouse heart using microarray assay and investigated their role in angiotensin II–induced cardiac remodeling. We found that let-7i was dynamically downregulated in angiotensin II–infused heart at day 3 and 7 and had the most targets that were mainly associated with cardiac inflammation and fibrosis. Overexpression or knockdown of let-7i in cultured cardiac fibroblasts demonstrated that let-7i played an inhibitory effect on the expression of its targets interleukin-6 and collagens. Furthermore, delivery of let-7i to mouse significantly inhibited angiotensin II–induced cardiac inflammation and fibrosis in a dose-dependent manner. Conversely, knockdown of let-7i aggravated this effect. Together, our results clearly demonstrate that let-7i acts as a novel negative regulator of angiotensin II–induced cardiac inflammation and fibrosis by suppressing the expression of interleukin-6 and multiple collagens in the heart and may represent a new potential therapeutic target for treating hypertensive cardiac fibrosis.

Baicalein Attenuates Angiotensin II-Induced Cardiac Remodeling via Inhibition of AKT/mTOR, ERK1/2, NF-κB, and Calcineurin Signaling Pathways in Mice

BACKGROUND Baicalein, a specific lipoxygenase (LOX) inhibitor, has anti-inflammatory and antioxidant effects. However, the functional role of baicalein in angiotensin II (Ang II)-induced hypertension and cardiac remodeling remains unclear. Here we investigated the effect of baicalein on cardiac hypertrophy and fibrosis and the underlying mechanism. METHODS Wild-type (WT) mice were injected with Ang II (1,200ng/kg/min) alone or together with 12/15-LOX inhibitor baicalein (25mg/kg) for 14 days. Histological examinations were performed on heart sections with hematoxylin and eosin, Massons trichrome, wheat germ agglutinin staining, and immunohistochemistry. The messenger RNA (mRNA) expression of cytokines and protein levels were detected by real-time polymerase chain reaction (PCR) and western blot analysis respectively. RESULTS Ang II infusion significantly increased blood pressure but decreased cardiac contractile function reflected by fractional shortening% and ejection fraction% compared with saline-treated mice. Moreover, Ang II infusion resulted in marked cardiac hypertrophy and fibrosis, promoted accumulation of macrophages and T cells, the expression of proinflammatory cytokines and malondialdehyde (MDA) production. However, these actions were markedly reversed by administration of baicalein in mice. Mechanistically, the protective effects of baicalein were associated with the inhibition of inflammation, oxidative stress, and multiple signaling pathways (AKT/mTOR, ERK1/2, nuclear factor-κB (NF-κB), and calcineurin) in the Ang II-treated mice. CONCLUSIONS This study demonstrates that baicalein can significantly ameliorate Ang II-induced hypertension and cardiac remodeling, and may be a novel therapeutic drug for prevention of hypertensive heart diseases.

Elevated expression of urotensin II and its receptor in skeletal muscle of diabetic mouse

Urotensin II (UII) is a somatostatin-like peptide recently identified to be involved in metabolic regulation and to play a significant role in diabetes and its complications. In the present study, we investigated the expression of UII and its receptor UT in the soleus muscle of male diabetic KK/upj-AY/J mice (2DM group) and the effects of UII on glucose uptake by the skeletal muscle to explore the role of skeletal muscle-derived UII in the pathogenesis of insulin resistance and diabetes. Radioimmunoassay, RT-PCR, immunohistochemistry and radio-ligand binding assay were used in this study. Compared with C57BL/6J mice (control group), 2DM mice showed increased UII content, by 34.0% in plasma, 15.4% in skeletal muscle tissue and 30.6% in medium containing UII from muscle (all P<0.05 or P<0.01). UII protein and UT mRNA expression were significantly enhanced in the skeletal muscle of 2DM mice. On [(125)I]UII binding to muscle sarcolemma, UT binding exhibited a saturable single-component characteristic in a specific and time-dependent manner. Scatchard plot analysis showed higher maximal number of specific binding sites (Bmax) in skeletal muscle, by 42.9% (P<0.01), and a lower dissociation constant (Kd), by 26.4% (P<0.01), in the 2DM group than in controls. On in vitro tissue pre-incubation with UII (10(-9), 10(-8) and 10(-7) mol/L), the insulin-stimulated [(3)H]-2-DG uptake by split soleus muscle was lower, by 9.5%, 33.4% and 39.7% (all P<0.01), respectively, than without UII incubation. UII/UT upregulated in skeletal muscle of 2DM mice suggests that UII derived from skeletal muscle might induce the pathogenesis of skeletal muscle insulin resistance as an autocrine factor.

A soluble receptor for advanced glycation end-products inhibits hypoxia/reoxygenation-induced apoptosis in rat cardiomyocytes via the mitochondrial pathway.

Severe myocardial dysfunction and tissue damage resulting from ischemia/reperfusion (I/R) is a common clinical scenario in patients with certain types of heart diseases and therapies such as thrombolysis, percutaneous coronary intervention, coronary artery bypass grafting, and cardiac transplantation. The underlining mechanism of endogenous cardiac protection after I/R injury has been a focus of current research. Growing evidences suggests that soluble receptor for advanced glycation end-products (sRAGE) has a cardioprotective effect; however, its role in I/R injury remains unclear. We hypothesized that exogenous administration of sRAGE during hypoxia/reoxygenation (H/R) induces cardioprotection by inhibiting cardiomyocyte apoptosis via multiple signals, involving mitochondrial membrane potential (MMP), the mitochondrial permeability transition pore (mPTP), mitochondrial cytochrome c, caspase-3, Bcl-2 and Bax. Neonatal rat cardiomyocytes underwent hypoxia for 3-h followed by 2-h reoxygenation or were treated with sRAGE for 10 min before H/R. Compared with H/R alone, sRAGE pretreatment reduced H/R-induced cardiomyocyte apoptosis from 27.9% ± 5.9% to 9.4% ± 0.7% (p < 0.05). In addition, sRAGE treatment significantly inhibited H/R-induced mitochondrial depolarization and mPTP opening, reduced mitochondrial cytochrome c leakage, caspase-3 and caspase-9 activity, and decreased the ratio of Bax to Bcl-2. Therefore, we conclude that the exogenous administration of sRAGE during H/R is involved in cardioprotection by inhibiting apoptosis via the mitochondrial pathway, which, if further confirmed in vivo, may have important clinical implications during H/R.

Angiotensin IV protects against angiotensin II-induced cardiac injury via AT4 receptor.

Angiotensin II (Ang II) is an important regulator of cardiac function and injury in hypertension. The novel Ang IV peptide/AT4 receptor system has been implicated in several physiological functions and has some effects opposite to those of Ang II. However, little is known about the role of this system in Ang II-induced cardiac injury. Here we studied the effect of Ang IV on Ang II-induced cardiac dysfunction and injury using isolated rat hearts, neonatal cardiomyocytes and cardiac fibroblasts. We found that Ang IV significantly improved Ang II-induced cardiac dysfunction and injury in the isolated heart in response to ischemia/reperfusion (I/R). Moreover, Ang IV inhibited Ang II-induced cardiac cell apoptosis, cardiomyocyte hypertrophy, and proliferation and collagen synthesis of cardiac fibroblasts; these effects were mediated through the AT4 receptor as confirmed by siRNA knockdown. These findings suggest that Ang IV may have a protective effect on Ang II-induced cardiac injury and dysfunction and may be a novel therapeutic target for hypertensive heart disease.

Identification of Genes Related to the Early Stage of Angiotensin II-induced Acute Renal Injury by Microarray and Integrated Gene Network Analysis

Background/Aims: Angiotensin II (Ang II) mediated signaling plays a key role in the development of chronic kidney damage that contributes to renal fibrosis. However, the gene expression changes regulated by Ang II in the early stage of acute renal injury remain unclear. Methods: C57BL/6 wild-type (WT) mice were injected with Ang II (1500 ng/kg/min) for 1, 3 and 7 days. A time series analysis of microarrays was performed to evaluate Ang II-induced differentially gene expression in the kidneys. The data of gene expression in the kidney was further dissected by ANOVA analysis, gene expression profiles, gene network construction and quantitative real-time RT-PCR. Ang II-induced renal inflammation and fibrosis in mice were confirmed by pathological examination. Results: Our microarray data showed that a total of 1,511 differentially expressed genes were identified in the kidneys at 1, 3 and 7 days after Ang II infusion. These genes function in multiple biological processes, including response to stimuli, immune response, cell adhesion, metabolic process, kidney development, regulation of blood pressure, and ion transport, which may play critical roles in the pathobiology of Ang II-induced acute renal injury at the early stage. Furthermore, among these genes, 20 genes were further selected for final investigation. The dynamic gene network analysis demonstrated that fatty acid binding protein 1 (Fabp1) localized in the core of the network. Conclusions: Our data suggests that genes involved in lipid metabolic process, especially Fabp1, may play a central role in the development of Ang II-induced acute renal injury at the early stage.