Establishment of a GFP::LMNB1 knockin cell line (CSUi002-A-1) from a dystonia patient-specific iPSC by CRISPR/Cas9 editing.

LMNB1, as one of the major components of nuclear lamina, anchors heterochromatin and associates with transcription regulation. LMNB1 was previously demonstrated to be upregulated and nuclear-to-cytoplasmic mislocalized in DYT1 dystonia specific neurons. Here, we established a knockin cell line with GFP::LMNB1 fusion expression from a DYT1 patient derived iPSC line, by CRISPR/Cas9 editing. The generated iPSCs displayed GFP and LMNB1 co-localization, reminiscent of successful genomic editing. They remained pluripotent and normal karyotype, and possessed the potential to differentiate into three germ layers. This GFP::LMNB1 knockin iPSC will be used for studying the lamina-pathophysiology of DYT1 dystonia, and other nucleus-centered questions.

miRNA-22 as a Candidate Diagnostic Biomarker for Coronary Slow Flow.

BACKGROUND: Coronary slow flow (CSF) refers to the phenomenon of delayed distal flow in the absence of lesions detected on coronary angiography. Although the detection rate of CSF has been increasing in clinical practice, early diagnosis is difficult and the factors contributing to this condition remain unclear. Given the increasing demonstration of the roles of microRNAs (miRNAs) in disease and as diagnostic biomarkers, the aim of this study was to analyze the expression of serum miRNA-22 in patients with CSF detected using coronary angiography and its diagnostic efficacy. METHODS AND RESULTS: A retrospective analysis including 44 patients with CSF and 42 patients with normal coronary flow (control group) was conducted. Additionally, all included patients either did not have visually estimated coronary artery stenosis or had <50% stenosis. Plasma samples were collected from patients in these two groups, and the levels of miRNA-22 were detected. The receiver operating characteristic (ROC) curve was plotted to evaluate the diagnostic efficiency of serum miRNA-22 in the context of CSF. RESULTS: The expression of serum miRNA-22 was significantly higher in the CSF patients than in the control subjects (P < 0.0001). The area under the ROC curve for miRNA-22 in diagnosing CSF was 0.8293 (95% confidence interval: 0.7313-0.9272), with a sensitivity of 75.0% and specificity of 88.1%. CONCLUSIONS: The expression of serum miRNA-22 in CSF is upregulated compared to that in subjects with normal coronary flow and shows relatively high clinical diagnostic efficiency, suggesting a new potential biomarker for the early diagnosis of CSF.

Plasma Choline as a Diagnostic Biomarker in Slow Coronary Flow.

AIM: The slow coronary flow (SCF) phenomenon was characterized by delayed perfusion of epicardial arteries, and no obvious coronary artery lesion in coronary angiography. The prognosis of patients with slow coronary flow was poor. However, there is lack of rapid, simple, and accurate method for SCF diagnosis. This study aimed to explore the utility of plasma choline as a diagnostic biomarker for SCF. METHODS: Patients with coronary artery stenosis <40% evaluated by the coronary angiogram method were recruited in this study and were grouped into normal coronary flow (NCF) and SCF by thrombolysis in myocardial infarction frame count (TFC). Plasma choline concentrations of patients with NCF and SCF were quantified by Ultra Performance Liquid Chromatography Tandem Mass Spectrometry. Correlation analysis was performed between plasma choline concentration and TFC. Receiver operating characteristic (ROC) curve analysis with or without confounding factor adjustment was applied to predict the diagnostic power of plasma choline in SCF. RESULTS: Forty-four patients with SCF and 21 patients with NCF were included in this study. TFC in LAD, LCX, and RCA and mean TFC were significantly higher in patients with SCF in comparison with patients with NCF (32.67 ± 8.37 vs. 20.66 ± 3.41, P < 0.01). Plasma choline level was obviously higher in patients with SCF when compared with patients with NCF (754.65 ± 238.18 vs. 635.79 ± 108.25, P=0.007). Plasma choline level had significantly positive correlation with Mean TFC (r = 0.364, P=0.002). Receiver operating characteristic (ROC) analysis showed that choline with or without confounding factor adjustment had an AUC score of 0.65 and 0.77, respectively. CONCLUSIONS: TFC were closely related with plasma choline level, and plasma choline can be a suitable and stable diagnostic biomarker for SCF.

Overexpression of long noncoding RNA ANRIL inhibits phenotypic switching of vascular smooth muscle cells to prevent atherosclerotic plaque development in vivo.

BACKGROUND: Phenotypic switching of vascular smooth muscle cells (VSMCs) plays a key role in atherosclerosis. Long noncoding RNA ANRIL (lncRNA-ANRIL) is critical in vascular homeostasis. Metformin produces multiple beneficial effects in atherosclerosis. However, the underlying mechanisms need to be elucidated. METHODS AND RESULTS: Metformin increased lncRNA-ANRIL expression and AMPK activity in cultured VSMCs, and inhibited the phenotypic switching of VSMCs to the synthetic phenotype induced by platelet-derived growth factor (PDGF). Overexpression of lncRNA-ANRIL inhibited phenotypic switching and reversed the reduction of AMPK activity in PDGF-treated VSMCs. While, gene knockdown of lncRNA-ANRIL by adenovirus or silence of AMPKγ through siRNA abolished AMPK activation induced by metformin in VSMCs. RNA-immunoprecipitation analysis indicated that the affinity of lncRNA-ANRIL to AMPKγ subunit was increased by metformin. In vivo, administration of metformin increased the levels of lncRNA-ANRIL, suppressed VSMC phenotypic switching, and prevented the development of atherosclerotic plaque in Apoe-/- mice fed with western diet. These protective effects of metformin were abolished by infecting Apoe-/- mice with adenovirus expressing lncRNA-ANRIL shRNA. The levels of AMPK phosphorylation, AMPK activity, and lncRNA-ANRIL expression were decreased in human atherosclerotic lesions. CONCLUSION: Metformin activates AMPK to suppress the formation of atherosclerotic plaque through upregulation of lncRNA-ANRIL.

NFAT5 promotes arteriogenesis via MCP-1-dependent monocyte recruitment.

Studies have demonstrated that nuclear factor of activated T cells 5 (NFAT5) is not only a tonicity-responsive transcription factor but also activated by other stimuli, so we aim to investigate whether NFAT5 participates in collateral arteries formation in rats. We performed femoral artery ligature (FAL) in rats for hindlimb ischaemia model and found that NFAT5 was up-regulated in rat adductors with FAL compared with sham group. Knockdown of NFAT5 with locally injection of adenovirus-mediated NFAT5-shRNA in rats significantly inhibited hindlimb blood perfusion recovery and arteriogenesis. Moreover, NFAT5 knockdown decreased macrophages infiltration and monocyte chemotactic protein-1 (MCP-1) expression in rats adductors. In vitro, with interleukin-1ß (IL-1ß) stimulation and loss-of-function studies, we demonstrated that NFAT5 knockdown inhibits MCP-1 expression in endothelial cells and chemotaxis of THP-1 cells regulated by ERK1/2 pathway. More importantly, exogenous MCP-1 delivery could recover hindlimb blood perfusion, promote arteriogenesis and macrophages infiltration in rats after FAL, which were depressed by NFAT5 knockdown. Besides, NFAT5 knockdown also inhibited angiogenesis in gastrocnemius muscles in rats. Our results indicate that NFAT5 is a critical regulator of arteriogenesis and angiogenesis via MCP-1-dependent monocyte recruitment, suggesting that NFAT5 may represent an alternative therapeutic target for ischaemic diseases.

Circulating miR-182-5p and miR-5187-5p as biomarkers for the diagnosis of unprotected left main coronary artery disease.

BACKGROUND: Patients with unprotected left main coronary artery disease (uLMCAD) have high mortality rate due to sudden heart failure and acute myocardial infarction, for which reliable diagnostic biomarkers to detect this disease at an early stage are in urgent need. Circulating microRNAs (miRNAs) have emerged as a class of novel biomarkers for cardiovascular diseases. The purpose of this study was to investigate utility of miRNAs as biomarkers for early detection of uLMCAD. METHODS: High-throughput sequencing (NGS) was initially employed to compare circulating miRNA expression profiles in uLMCAD patients to that in patients without coronary artery disease (CAD) to identify candidate miRNA biomarkers. We further validated the expression of candidate miRNAs by quantitative polymerase chain reaction (qPCR) in a larger cohort. Receiver operating characteristic (ROC) analysis with multivariate logistic regression was used to evaluate the diagnostic power of candidate miRNAs individually and combined. RESULTS: MiR-182-5p, miR-199a-5p and miR-5187-5p were found significantly differentially expressed through NGS (fold changes =1.35, 1.65, 0.5, P values =0.018, 0.046, 0.030, respectively, n=5 for both uLMCAD group and non-CAD control group). In a larger cohort (n=27 for uLMCAD patient and n=38 for non-CAD controls), qPCR confirmed that expression of miR-182-5p was up-regulated (2.57-fold, P=0.011) and expression of miR-5187-5p was down-regulated (0.47-fold, P=0.018) in the plasma of uLMCAD patients. ROC analysis with multivariate logistic regression show that miR-182 and miR-5187 have an AUC score of 0.97 and 0.94 respectively, indicating high diagnostic power as biomarkers for uLMCAD. Interestingly, correlation analysis suggests that the expression of two miRNAs were independent to each other. CONCLUSIONS: These results suggested that circulating miR-182-5p and miR-5187-5p were suitable diagnostic biomarkers for uLMCAD, both potentially providing diagnostic information for discriminating uLMCAD patients from non-CAD population prior to invasive diagnostic coronary angiography (CAG).

Endothelial progenitor cell impairment mediated vasodilation dysfunction via diminishing nitric oxide production in postmenopausal females.

Vascular endothelial dysfunction is the major contributing factor to hypertension. Endothelial progenitor cells (EPCs) are essential for endogenous vascular endothelial renovation. The activity and number of circulating EPCs are preserved in prehypertensive premenopausal females according to our previous research. However, the changes of EPCs in prehypertensive postmenopausal females are poorly understood, and the mechanisms responsible for the loss of the gender protection advantage of cardiovascular disease remain unexplored. In order to determine the effects of EPCs in prehypertensive postmenopausal females, the number and activity of circulating EPCs were tested in the present study. Next, the function of EPCs secreting nitric oxide (NO), vascular endothelial growth factor (VEGF) and granulocyte­macrophage colony­stimulating factor (GM­CSF), as well as their concentration in the plasma, were measured. The association between flow­mediated dilation (FMD) and EPC secretion was also assessed. Attenuation of proliferation and migration of EPCs was observed in prehypertensive patients in comparison with normotensive subjects. In addition, a reduced NO production secreted by EPCs was detected in prehypertensive patients as compared with that in normotensive patients. There was no significant difference in EPC function between postmenopausal females and age­matched males. Finally, the association between FMD and NO production was validated. Collectively, these data indicated that impaired EPCs mediated vasodilation dysfunction via decreasing NO production. Therefore, EPC function enhancement and NO level augmentation are emerging as novel therapeutic strategies for prehypertension therapy.

Human tissue kallikrein-1 protects against the development of erectile dysfunction in a rat model of hyperhomocysteinemia.

The aim of this study was to investigate the mechanism by which a diet inducing high hyperhomocysteinemia (HHcy) leads to the deterioration of erectile function in rats and whether this is inhibited by expression of the human tissue kallikrein-1 (hKLK1) gene. We established a rat model of HHcy by feeding methionine (Met)-rich diets to male Sprague-Dawley (SD) rats. Male wild-type SD rats (WTRs) and transgenic rats harboring the hKLK1 gene (TGRs) were fed a normal diet until 10 weeks of age. Then, 30 WTRs were randomly divided into three groups as follows: the control (n = 10) group, the low-dose (4% Met, n = 10) group, and the high-dose (7% Met, n = 10) group. Another 10 age-matched TGRs were fed the high-dose diet and designated as the TGR+7% Met group. After 30 days, in all four groups, erectile function was measured and penile tissues were harvested to determine oxidative stress, endothelial cell content, and penis fibrosis. Compared with the 7% Met group, the TGR+7% Met group showed diminished HHcy-induced erectile dysfunction (ED), indicating the improvement caused by hKLK1. Regarding corpus cavernosum endothelial cells, hKLK1 preserved endothelial cell-cell junctions and endothelial cell content, and activated protein kinase B/endothelial nitric oxide synthase (Akt/eNOS) signaling. Fibrosis assessment indicated that hKLK1 preserved normal penis structure by inhibiting apoptosis in the corpus cavernosum smooth muscle cells. Taken together, these findings showed that oxidative stress, impaired corpus cavernosum endothelial cells, and severe penis fibrosis were involved in the induction of ED by HHcy in rats, whereas hKLK1 preserved erectile function by inhibiting these pathophysiological changes.

Endogenous reduction of miR-185 accelerates cardiac function recovery in mice following myocardial infarction via targeting of cathepsin K.

Angiogenesis is critical for re-establishing the blood supply to the surviving myocardium after myocardial infarction (MI) in patients with acute coronary syndrome (ACS). MicroRNAs are recognised as important epigenetic regulators of endothelial function. The aim of this study was to determine the roles of microRNAs in angiogenesis. Eighteen circulating microRNAs including miR-185-5p were differently expressed in plasma from patients with ACS by high-throughput RNA sequencing. The expressional levels of miR-185-5p were dramatically reduced in hearts isolated from mice following MI and cultured human umbilical vein endothelial cells (HUVECs) under hypoxia, as determined by fluorescence in situ hybridisation and quantitative RT-PCR. Evidence from computational prediction and luciferase reporter gene activity indicated that cathepsin K (CatK) mRNA is a target of miR-185-5p. In HUVECs, miR-185-5p mimics inhibited cell proliferations, migrations and tube formations under hypoxia, while miR-185-5p inhibitors performed the opposites. Further, the inhibitory effects of miR-185-5p up-regulation on cellular functions of HUVECs were abolished by CatK gene overexpression, and adenovirus-mediated CatK gene silencing ablated these enhancive effects in HUVECs under hypoxia. In vivo studies indicated that gain-function of miR-185-5p by agomir infusion down-regulated CatK gene expression, impaired angiogenesis and delayed the recovery of cardiac functions in mice following MI. These actions of miR-185-5p agonists were mirrored by in vivo knockdown of CatK in mice with MI. Endogenous reductions of miR-185-5p in endothelial cells induced by hypoxia increase CatK gene expression to promote angiogenesis and to accelerate the recovery of cardiac function in mice following MI.

Hypoxia-elicited mesenchymal stem cell-derived exosomes facilitates cardiac repair through miR-125b-mediated prevention of cell death in myocardial infarction.

Exosomes (Exo) secreted from hypoxia-conditioned bone marrow mesenchymal stem cells (BM-MSCs) were found to be protective for ischemic disease. However, the role of exosomal miRNA in the protective effect of hypoxia-conditioned BM-MSCs-derived Exo (Hypo-Exo) remains largely uncharacterized and the poor specificity of tissue targeting of Exo limits their clinical applications. Therefore, the objective of this study was to examine the effect of miRNA in Hypo-Exo on the repair of ischemic myocardium and its underlying mechanisms. We further developed modified Hypo-Exo with high specificity to the myocardium and evaluate its therapeutic effects. Methods: Murine BM-MSCs were subjected to hypoxia or normoxia culture and Exo were subsequently collected. Hypo-Exo or normoxia-conditioned BM-MSC-derived Exo (Nor-Exo) were administered to mice with permanent condition of myocardial infarction (MI). After 28 days, to evaluate the therapeutic effects of Hypo-Exo, infarction area and cardio output in Hypo-Exo and Nor-Exo treated MI mice were compared through Masson's trichrome staining and echocardiography respectively. We utilized the miRNA array to identify the significantly differentially expressed miRNAs between Nor-Exo and Hypo-Exo. One of the most enriched miRNA in Hypo-Exo was knockdown by applying antimiR in Hypoxia-conditioned BM-MSCs. Then we performed intramyocardial injection of candidate miRNA-knockdown-Hypo-Exo in a murine MI model, changes in the candidate miRNA's targets expression of cardiomyocytes and the cardiac function were characterized. We conjugated Hypo-Exo with an ischemic myocardium-targeted (IMT) peptide by bio-orthogonal chemistry, and tested its targeting specificity and therapeutic efficiency via systemic administration in the MI mice. Results: The miRNA array revealed significant enrichment of miR-125b-5p in Hypo-Exo compared with Nor-Exo. Administration of miR-125b knockdown Hypo-Exo significantly increased the infarction area and suppressed cardiomyocyte survival post-MI. Mechanistically, miR-125b knockdown Hypo-Exo lost the capability to suppress the expression of the proapoptotic genes p53 and BAK1 in cardiomyocytes. Intravenous administration of IMT-conjugated Hypo-Exo (IMT-Exo) showed specific targeting to the ischemic lesions in the injured heart and exerted a marked cardioprotective function post-MI. Conclusion: Our results illustrate a new mechanism by which Hypo-Exo-derived miR125b-5p facilitates ischemic cardiac repair by ameliorating cardiomyocyte apoptosis. Furthermore, our IMT- Exo may serve as a novel drug carrier that enhances the specificity of drug delivery for ischemic disease.

Lipoxin A4 improves erectile dysfunction in rats with type I diabetes by inhibiting oxidative stress and corporal fibrosis.

Previous studies have shown that oxidative stress and corporal fibrosis in penile tissues of rats were key pathological factors of erectile dysfunction induced by diabetic mellitus (DMED). Lipoxin A4 (LXA4) was reported to inhibit oxidative stress and fibrosis diseases, while whether it could exert a protective role on erectile function was not clear. Type I diabetic mellitus (DM) was induced in thirty male 10-week-old Sprague-Dawley rats using streptozotocin. Ten weeks later, twenty-two rats with DMED confirmed by an apomorphine test were divided into two groups: the DMED group (n = 11) and the DMED + LXA4 group (n = 11; LXA4 injection daily for 4 weeks). In addition, another ten age-matched rats formed the Control group. We found that erectile function was significantly impaired in the DMED group compared with the Control group, but was improved in the DMED + LXA4 group. Similarly, the over-activated oxidative stress and impaired endothelial function in the DMED group were both improved in the DMED + LXA4 group. Moreover, the DMED group showed serious corporal fibrosis, which was also inhibited by the treatment of LXA4 in the DMED + LXA4 group. Taken together, LXA4 could exert an inhibition role on oxidative stress and fibrosis to improve DMED effectively.

MiR-15b-5p Regulates Collateral Artery Formation by Targeting AKT3 (Protein Kinase B-3).

OBJECTIVE: To identify circulating microRNAs that are differentially expressed in severe coronary heart disease with well or poorly developed collateral arteries and to investigate their mechanisms of action in vivo and in vitro. APPROACH AND RESULTS: In our study, we identified a circulating microRNA, miR-15b-5p, with low expression that, nevertheless, characterized patients with sufficient coronary collateral artery function. Moreover, in murine hindlimb ischemia model, in situ hybridization identified that miR-15b-5p was specifically expressed in vascular endothelial cells of adductors in sham group and was remarkably downregulated after femoral artery ligation. Overexpressed miR-15b-5p significantly inhibited arteriogenesis and angiogenesis in mice. In vitro, both under basal and vascular endothelial growth factor stimulation, loss-of-function or gain-of-function studies suggested that miR-15b-5p significantly promoted or depressed the migration and proliferation of endothelial cells. We identified AKT3 (protein kinase B-3) as a direct target of miR-15b-5p. Interestingly, AKT3 deficiency by injection with Chol-AKT3-siRNA obviously suppressed arteriogenesis and the recovery of blood perfusion after femoral ligation in mice. CONCLUSIONS: These results indicate that circulating miR-15b-5p is a suitable biomarker for discriminating between patients with well-developed or poorly developed collaterals. Moreover, miR-15b-5p is a key regulator of arteriogenesis and angiogenesis, which may represent a potential therapeutic target for ischemic disease.

Plasma Omentin-1 Level as a Predictor of Good Coronary Collateral Circulation.

AIM: Coronary collateral circulation (CCC) is crucial during an acute ischemic attack. Evidences showed that omentin-1 exhibited remarkable antiatherogenic effects and ischemia-induced revascularization. The aim of this study was to investigate the relationship between plasma omentin-1 levels and CCC in patients with ≥90% angiography-proven coronary occlusion. METHODS: 142 patients with ≥90% luminal diameter stenosis in at least one major epicardial coronary artery were recruited. Among them, 79 patients with Rentrop 0-1 grade were classified into the poor CCC group and 63 patients with Rentrop 2-3 grade were included into the good CCC group. The association between plasma omentin-1 levels and CCC status was assessed. RESULTS: Plasma omentin-1 level was significantly higher in patients with good CCC than those with poor CCC (566.57±26.90 vs. 492.38±19.70 ng/mL, p=0.024). Besides, omentin-1 was positively correlated with total cholesterol (TC), high-density lipoprotein, and gensini score but inversely with hyperlipidemia and body mass index (all p values＜0.05). Multivariate regression analysis indicated that omentin-1 [odds ratio (OR)=1.002, 95% confidence interval (CI): 1.000-1.004, p=0.041)], TC, the number of the diseased vessels, a higher frequency of left circumflex artery and right coronary artery, chronic total occlusion, and gensini score remained as the independent predictors of good CCC. CONCLUSION: Higher plasma omentin-1 level was associated with better CCC development. Our findings suggest that omentin-1 may be an alternative marker for adequate CCC in patients with ≥90% coronary occlusion.

High-Density Lipoprotein Prevents Endoplasmic Reticulum Stress-Induced Downregulation of Liver LOX-1 Expression.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a specific cell-surface receptor for oxidized-low-density lipoprotein (ox-LDL). The impact of high-density lipoprotein (HDL) on endoplasmic reticulum (ER) stress-mediated alteration of the LOX-1 level in hepatocytes remains unclear. We aimed to investigate the impact on LOX-1 expression by tunicamycin (TM)-induced ER stress and to determine the effect of HDL on TM-affected LOX-1 expression in hepatic L02 cells. Overexpression or silencing of related cellular genes was conducted in TM-treated cells. mRNA expression was evaluated using real-time polymerase chain reaction (PCR). Protein expression was analyzed by western blot and immunocytochemistry. Lipid uptake was examined by DiI-ox-LDL, followed by flow cytometric analysis. The results showed that TM induced the upregulation of ER chaperone GRP78, downregulation of LOX-1 expression, and lipid uptake. Knock down of IRE1 or XBP-1 effectively restored LOX-1 expression and improved lipid uptake in TM-treated cells. HDL treatment prevented the negative impact on LOX-1 expression and lipid uptake induced by TM. Additionally, 1-10 µg/mL HDL significantly reduced the GRP78, IRE1, and XBP-1 expression levels in TM-treated cells. Our findings reveal that HDL could prevent the TM-induced reduction of LOX-1 expression via inhibiting the IRE1/XBP-1 pathway, suggesting a new mechanism for beneficial roles of HDL in improving lipid metabolism.

Asymmetric dimethylarginine triggers macrophage apoptosis via the endoplasmic reticulum stress pathway.

Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), is emerging as a key contributing factor in atherogenesis, a process in turn known to involve macrophage apoptosis. The aim of this study was to determine the effect of ADMA on macrophage apoptosis, with specific reference to the endoplasmic reticulum (ER) stress pathway. Macrophage apoptosis was evaluated by Annexin V- Propidium iodide (PI) and Hoechst 33258 staining assays. Levels of the ER stress marker glucose regulated protein 78 (GRP78) were characterized by western blot. Levels of the proapoptotic C/EBP-homologous protein (CHOP) were evaluated by western blot and reverse transcription polymerase chain reaction (RT-PCR), and caspase-4 activity was measured using a colorimetric protease assay kit. We observed ADMA dose- and time-dependent increases in macrophage levels of GRP78. Similar ADMA dose- and time-dependent increases were detected in intracellular caspase-4 activity and macrophage apoptosis, all of which were sensitive to treatment with siRNAs for protein kinase RNA-like ER kinase and inositol-requiring protein-1 (IRE1), the ADMA antagonist L-arginine, as well as inhibitors of eukaryotic translation initiation factor-2 (salubrinal), IRE1 (irestatin 9389), and c-Jun N-terminal kinase (SP600125). Our results indicate that ADMA triggers macrophage apoptosis via the ER stress pathway.

Ang II induces capillary formation from endothelial cells via the AT1R-dependent inositol requiring enzyme 1 pathway.

Previous studies have demonstrated an important interaction between angiotension II type 1 receptor (AT1R) and angiotension II (Ang II) -induced capillary formation from endothelial cells and vascular endothelial growth factor (VEGF). However, the underlying mechanism remains elusive. Recent studies revealed that the unfolded protein response regulates an angiogenic response by the kidney epithelium during ischemic stress. Therefore, in the present study, we investigated the effects of Ang II on AT1R-mediated capillary formation from endothelial cells and the possible involvement of the IRE1/JNK/p38 MAPK pathway. Our results show that Ang II (1 nmol/L) induced the expression of VEGF and enhanced capillary formation from endothelial cells in the Matrigel assay. This effect was significantly depressed by the AT1R blocker losartan and different inhibitors (irestatin, IRE1 specific inhibitor; SP600125, JNK specific inhibitor; SB203580, p38 MAPK specific inhibitor) but not by the AT2R blocker PD123319. Next, we investigated the effect of Ang II on the IRE1/JNK/p38 MAPK pathway and the 78kDA glucose regulated protein 78 (GRP78) activity in HUVECs and the role of the AT1 Receptor. The results show that Ang II activated both the IRE1/JNK/p38 MAPK pathway and GRP78 binding activity. These effects were markedly inhibited by the AT1R blocker losartan. The IRE1 specific inhibitor irestatin, the JNK specific inhibitor SP600125, and the p38 MAPK specific inhibitor SB203580 significantly inhibited Ang II-induced capillary formation from endothelial cells and VEGF expression but had no effect on GRP78. Collectively, these findings suggest for the first time that Ang II promotes capillary formation by inducing the expression of VEGF via Ang II type 1 receptor-mediated stimulation of the IRE1/JNK/p38 MAPK pathway.

Influence of ALDH2 Glu504Lys polymorphism on nitroglycerin response in chronic heart failure and involvement of Calcitonin Gene Related Peptide (CGRP).

OBJECTIVE: The aim of this study was to investigate whether the neuropeptide calcitonin gene related peptide (CGRP) contributes to nitroglycerin (GTN) response in patients with chronic heart failure (CHF) and the association with the mitochondrial aldehyde dehydrogenase-2 (ALDH2) Glu504Lys (ALDH2*2) polymorphism. METHODS: This is a 2-period, placebo-controlled clinical study. An intravenous infusion of saline followed by GTN (20 µg/min), each for 2 hours, respectively, was given to 49 stable CHF patients. Blood pressure (BP), heart rate (HR) and respiratory rate (RR) were measured at baseline, at 10 min, 30 min, 1.0 h, 1.5 h, and 2.0 h after initiation of saline infusion and initiation of GTN therapy. Blood samples were drawn for the determination of plasma CGRP for 49 patients at baseline, and at 2.0 h after initiation of saline and GTN infusion, respectively. Global clinical status of the patients was evaluated. Left ventricular ejection (LVEF), left ventricular end-diastolic volume (LVEDV), stroke volume (SV) and cardiac output (CO) were measured with 2D echocardiography with Simpson's biplane method (Pillip HP sonos 5500) by the same investigator at baseline and at 2.0 h after initiation of saline and GTN infusion. RESULTS: Systolic blood pressure (SBP), diastolic blood pressure (DBP), and left ventricular end-diastolic volume (LVEDV) were decreased, while left ventricular ejection fraction (LVEF) was increased at the end of GTN infusion (p < 0.001, respectively). Saline infusion showed no hemodynamic effects. At the end of GTN infusion, ALDH2*1/*1 homozygous patients showed higher degrees of both the absolute decrease in SBP (DSBP) (p < 0.001) and increase in LVEF (p < 0.001) than carriers of the ALDH2*2 allele. Mean plasma concentration of CGRP was increased after GTN infusion (p < 0.001), but not changed after saline infusion (p > 0.05). Changes in plasma concentration of CGRP correlated positively with the improvement in LVEF (r = 0.400, p = 0.004), while correlated negatively with changes in SBP (r = -0.300, p = 0.036) and LVEDV (r = -0.290, p = 0.043). CONCLUSIONS: ALDH2*2 polymorphism is associated with contributions of CGRP to GTN response in CHF patients.

[Transthoracic echocardiography in transcatheter closure of atrial septal aneurysm combined with secoundum-type atrial septal defect].

OBJECTIVE: To explore the value of transthoracic echocardiography (TTE) in transcatheter closure of atrial septal aneurysm (ASA) combined with secoundum-type atrial septal defect (ASD). METHODS: Fourteen patients (3 males and 11 females) who had ASA combined with secoundum-type ASD were diagnosed by TTE or transesophageal echocardiography. The ASA projected to the right atrium in all patients. The width of basilar part was 13 approximately 24 (18.5+/-3.9) mm, and the vertical extent was 7 approximately 11(9.7+/-1.8) mm. Ten patients combined with single hole ASD and 4 patients with multiple hole ASD. Blood shifting from the left atrium to the right atrium was displayed in color Doppler in all patients. All patients were treated by transcatheter closure under the guiding of X fluoroscopy and TTE, and examined with TTE during the follow-up. RESULTS: Transcatheter closure was successfully performed by 14 occluders in all patients. No residual shunt was detected immediately by TTE after the procedure in all patients. During the 6 approximately 12 month follow-up, no residual shunt or occluder shifting was found, the dimensions of the heart became normal in 11 patients (79%) and were significantly decreased in 4. CONCLUSION: Transcatheter closure is feasible in patients with ASA combined with secoundum-type ASD, and extra attention must be paid to the specialty. TTE is very important in case selection before transcatheter closure, and it may be used to monitor and guide the procedure during transcatheter closure.

[Expression of cardiotrophin-1 and effects of irbesartan in adriamycin induced cardiomyopathy in rats].

OBJECTIVE: To investigate cardiotrophin-1(CT-1) expression in the ventricle and the effects of angiotensin II type I receptor antagonist (AT(1)RA) irbesartan on the ventricular remodeling in adriamycin myocardiopathy. METHODS: Thirty male SD rats were randomized into 2 groups: a control group (n=10) and a model group (n=20). The model group was administered adriamycin and 18 rats survived. And theses rats were randomized again into 2 groups. One was treated with irbesartan [50 mg/(kg x d), with stomach-tube], and the other received equal saline, so did the control group. After 12 weeks, the protein level of CT-1 was detected by immunohistochemistry. RESULTS: Ventricular CT-1 in the model control group and the treatment group was higher than that in the control group and the correlation analysis showed that ventricular CT-1 of the model control group was positively correlated with the left ventricular weight index, and CT-1 of the treatment group was lower than that of the model control group. CONCLUSION: CT-1 was assumed to take part in the ventricular remodeling. The mechanism of irbesartan on the ventricular remodeling may be related to the downregulation of CT-1 expression.

[Short and mid-term effects of percutaneous transcatheter closure of ventricular septal defects on the cardiac remodeling].

OBJECTIVE: To evaluate the short and mid-term changes of the cardiac morphology after percutaneous transcatheter closure of ventricular septal defects (VSD) with transthoracic echocardiography (TTE). METHODS: The left ventricular end-diastolic diameter (LVEDD), left ventricular end-diastolic volume (LVEDV), left atrial diameter (LAd), and right ventricular diameter (RVd) in 30 VSD patients were measured before the VSD closure,and on the 3rd day, 3rd month, and 6th month after the VSD closure by TTE. RESULTS: LVEDD and LVEDV significantly decreased on the 3rd day after the VSD closure compared with pre-VSD closure. LVEDD and LVEDV continuously decreased on the 3rd month and 6th month after the VSD closure. LAd was smaller on the 3rd month and 6th month after the VSD closure, but there was not significant difference between the 3rd and 6th month. RVd increased on the 3rd day after the VSD closure, while no significant difference was found among the 3rd month and 6th month before and after VSD closure. CONCLUSION: Percutaneous transcatheter VSD closure may effectively improve the cardiac remodeling in VSD patients in the short and mid-term follow-up.