Naifeng Liu

Clinical Implication of Coronary Tortuosity in Patients with Coronary Artery Disease

Background Coronary tortuosity (CT) is a common coronary angiography finding. The exact pathogenesis, clinical implication and long-term prognosis of CT are not fully understood. The purpose of this study is to investigate the clinical characteristics of CT in patients with suspected coronary artery disease(CAD) in a Chinese population. Methods A total of 1010 consecutive patients underwent coronary angiography with complaints of chest pain or related symptoms were included in the present study (544 male, mean age: 64±11 years). CT was defined by the finding of ≥3 bends (defined as ≥45° change in vessel direction) along main trunk of at least one artery in systole and in diastole. Patients with or without CAD were further divided into CT-positive and CT-negative groups, all patients were followed up for the incidence of major adverse cardiovascular events (MACE) for 2 to 4 years. Results The prevalence of CT was 39.1% in this patient cohort and incidence of CT was significantly higher in female patients than that in male patients (OR = 2.603, 95%CI 1.897, 3.607, P<0.001). CT was positively correlated with essential hypertension (OR = 1.533, 95%CI 1.131, 2.076, P = 0.006) and negatively correlated with CAD (OR = 0.755, 95%CI 0.574, 0.994, P = 0.045). MACE during follow up was similar between CAD patients with or without CT. Conclusions CT is more often seen in females and positively correlated with hypertension and negatively correlated with coronary atherosclerosis.

Erythropoietin attenuates cardiac dysfunction by increasing myocardial angiogenesis and inhibiting interstitial fibrosis in diabetic rats

BackgroundRecent studies revealed that erythropoietin (EPO) has tissue-protective effects in the heart by increasing vascular endothelial growth factor (VEGF) expression and attenuating myocardial fibrosis in ischemia models. In this study, we investigated the effect of EPO on ventricular remodeling and blood vessel growth in diabetic rats.MethodsMale SD rats were randomly divided into 3 groups: control rats, streptozotocin (STZ)-induced diabetic rats, and diabetic rats treated with 1000 U/kg EPO by subcutaneous injection once per week. Twelve weeks later, echocardiography was conducted, and blood samples were collected for counting of peripheral blood endothelial progenitor cells (EPCs). Myocardial tissues were collected, quantitative real-time PCR (RT-PCR) was used to detect the mRNA expression of VEGF and EPO-receptor (EPOR), and Western blotting was used to detect the protein expression of VEGF and EPOR. VEGF, EPOR, transforming growth factor beta (TGF-β), and CD31 levels in the myocardium were determined by immunohistochemistry. To detect cardiac hypertrophy, immunohistochemistry of collagen type I, collagen type III, and Picrosirius Red staining were performed, and cardiomyocyte cross-sectional area was measured.ResultsAfter 12 weeks STZ injection, blood glucose increased significantly and remained consistently elevated. EPO treatment significantly improved cardiac contractility and reduced diastolic dysfunction. Rats receiving the EPO injection showed a significant increase in circulating EPCs (27.85 ± 3.43%, P < 0.01) compared with diabetic untreated animals. EPO injection significantly increased capillary density as well as EPOR and VEGF expression in left ventricular myocardial tissue from diabetic rats. Moreover, EPO inhibited interstitial collagen deposition and reduced TGF-β expression.ConclusionsTreatment with EPO protects cardiac tissue in diabetic animals by increasing VEGF and EPOR expression levels, leading to improved revascularization and the inhibition of cardiac fibrosis.

Advanced glycation endproducts increase EPC apoptosis and decrease nitric oxide release via MAPK pathways.

OBJECTIVE Previous studies have shown that advanced glycation endproducts (AGE) can induce endothelial progenitor cells (EPC) apoptosis, which contributes to the pathogenesis of diabetes mellitus. Nitric oxide (NO) signaling is closely associated with apoptosis. We therefore investigated the effects of AGE on human EPC apoptosis, NO release and related signal transduction pathways. METHODS EPC isolated from healthy human subjects were cultured with various concentrations of AGE (0, 2, 20 and 200mg/L) for 0, 24, 48 and 72 h in the presence or absence of various MAPK (ERK/P38/JNK) inhibitors, respectively. EPC apoptosis (detected by flow cytometric analyses) and NO concentration in culture supernatant were determined. The mRNA levels of eNOS, COX-2, Bcl-2 and Bax were assessed by RT-PCR and the protein expressions of NF-kappaB and Caspase-3 assessed by Western blot. RESULTS Increased EPC apoptosis and reduced NO release were induced by 200mg/L AGE, accompanied by a downregulation of eNOS and Bcl-2 expressions as well as an elevation in COX-2, Bax, NF-kappaB and Caspase-3 expressions in a time-dependent manner (all P<0.05). These changes were significantly attenuated by pretreatment with various MAPK (ERK/P38/JNK) inhibitors (P<0.05). CONCLUSIONS AGE can promote EPC apoptosis and decrease NO release via MAPK pathways.

Tissue Kallikrein Promotes Cardiac Neovascularization by Enhancing Endothelial Progenitor Cell Functional Capacity

Tissue kallikrein (TK) has been demonstrated to improve neovasculogenesis after myocardial infarction (MI). In the present study, we examined the role and underlying mechanisms of TK in peripheral endothelial progenitor cell (EPC) function. Peripheral blood-derived mononuclear cells containing EPCs were isolated from rat. The in vitro effects of TK on EPC differentiation, apoptosis, migration, and vascular tube formation capacity were studied in the presence or absence of TK, kinin B(2) receptor antagonist (icatibant), and phosphatidylinositol-3 kinase inhibitor (LY294002). Apoptosis was evaluated by flow-cytometry analysis using Annexin V-FITC/PI staining, as well as western-blot analysis of Akt phosphorylation and cleaved caspase-3. Using an MI mouse model, we then examined the in vivo effects of human TK gene adenoviral vector (Ad.hTK) administration on the number of CD34(+)Flk-1(+) progenitors in the peripheral circulation, heart tissue, extent of vasculogenesis, and heart function. Administration of TK significantly increased the number of Dil-LDL/UEA-lectin double-positive early EPCs, as well as their migration and tube formation properties in vitro. Transduction of TK in cultured EPCs attenuated apoptosis induced by hypoxia and led to an increase in Akt phosphorylation and a decrease in cleaved caspase-3 levels. The beneficial effects of TK were blocked by pretreatment with icatibant and LY294002. The expression of recombinant human TK in the ischemic mouse heart significantly improved cardiac contractility and reduced infarct size 7 days after gene delivery. Compared with the Ad.Null group, Ad.hTK reduced mortality and preserved left ventricular function by increasing the number of CD34(+)Flk-1(+) EPCs and promoting the growth of capillaries and arterioles in the peri-infarct myocardium. These data provide direct evidence that TK promotes vessel growth by increasing the number of EPCs and enhancing their functional properties through the kinin B(2) receptor-Akt signaling pathway.

Impact of coronary tortuosity on coronary pressure: numerical simulation study.

Background Coronary tortuosity (CT) is a common coronary angiographic finding. Whether CT leads to an apparent reduction in coronary pressure distal to the tortuous segment of the coronary artery is still unknown. The purpose of this study is to determine the impact of CT on coronary pressure distribution by numerical simulation. Methods 21 idealized models were created to investigate the influence of coronary tortuosity angle (CTA) and coronary tortuosity number (CTN) on coronary pressure distribution. A 2D incompressible Newtonian flow was assumed and the computational simulation was performed using finite volume method. CTA of 30°, 60°, 90°, 120° and CTN of 0, 1, 2, 3, 4, 5 were discussed under both steady and pulsatile conditions, and the changes of outlet pressure and inlet velocity during the cardiac cycle were considered. Results Coronary pressure distribution was affected both by CTA and CTN. We found that the pressure drop between the start and the end of the CT segment decreased with CTA, and the length of the CT segment also declined with CTA. An increase in CTN resulted in an increase in the pressure drop. Conclusions Compared to no-CT, CT can results in more decrease of coronary blood pressure in dependence on the severity of tortuosity and severe CT may cause myocardial ischemia.

In Vivo Imaging of Macrophages during the Early-Stages of Abdominal Aortic Aneurysm Using High Resolution MRI in ApoE−/− Mice

Background Angiotensin II (ANG II) promotes vascular inflammation and induces abdominal aortic aneurysm (AAA) in hyperlipidemic apolipoprotein E knock-out (apoE−/−) mice. The aim of the present study was to detect macrophage activities in an ANG II-induced early-stage AAA model using superparamagnetic iron oxide (SPIO) as a marker. Methodology/Principal Findings Twenty-six male apoE−/− mice received saline or ANG II (1000 or 500 ng/kg/min) infusion for 14 days. All animals underwent MRI scanning following administration of SPIO with the exception of three mice in the 1000 ng ANG II group, which were scanned without SPIO administration. MR imaging was performed using black-blood T2 to proton density -weighted multi-spin multi-echo sequence. In vivo MRI measurement of SPIO uptake and abdominal aortic diameter were obtained. Prussian blue, CD68,α-SMC and MAC3 immunohistological stains were used for the detection of SPIO, macrophages and smooth muscle cells. ANG II infusion with 1000 ng/kg/min induced AAA in all of the apoE−/− mice. ANG II infusion exhibited significantly higher degrees of SPIO uptake, which was detected using MRI as a distinct loss of signal intensity. The contrast-to-noise ratio value decreased in proportion to an increase in the number of iron-laden macrophages in the aneurysm. The aneurysmal vessel wall in both groups of ANG II treated mice contained more iron-positive macrophages than saline-treated mice. However, the presence of cells capable of phagocytosing haemosiderin in mural thrombi also induced low-signal-intensities via MRI imaging. Conclusions/Significance SPIO is taken up by macrophages in the shoulder and the outer layer of AAA. This alters the MRI signaling properties and can be used in imaging inflammation associated with AAA. It is important to compare images of the aorta before and after SPIO injection.

Advanced glycation end-products decreases expression of endothelial nitric oxide synthase through oxidative stress in human coronary artery endothelial cells

BackgroundAdvanced glycation end-products (AGEs) are elevated under diabetic conditions and associated with insulin resistance, endothelial dysfunction and vascular inflammation in humans. It has been demonstrated that AGEs evoke oxidative and inflammatory reactions in endothelial cells through the interaction with a receptor for AGEs (RAGE). Here, we aimed to identify the cellular mechanisms by which AGEs exacerbate the endothelial dysfunction in human coronary artery endothelial cells (HCAECs).Methods30 type 2 diabetic patients with or without coronary artery atherosclerosis were recruited for this study. Plasma levels of AGE peptides (AGE-p) were analyzed using flow injection assay. Endothelial function was tested by brachial artery flow-mediated vasodilatation (FMD). Further investigations were performed to determine the effects and mechanisms of AGEs on endothelial dysfunction in HCAECs.ResultsAGE-p was inversely associated with FMD in diabetic patients with coronary artery atherosclerosis in our study. After treated with AGEs, HCAECs showed significant reductions of eNOS mRNA and protein levels including eNOS and phospho-eNOS Ser1177, eNOS mRNA stability, eNOS enzyme activity, and cellular nitric oxide (NO) levels, whereas superoxide anion production was significantly increased. In addition, AGEs significantly decreased mitochondrial membrane potential, ATP content and catalase and superoxyde dismutase (SOD) activities, whereas it increased NADPH oxidase activity. Treatment of the cells with antioxidants SeMet, SOD mimetic MnTBAP and mitochondrial inhibitor thenoyltrifluoroacetone (TTFA) effectively blocked these effects induced by AGEs. AGEs also increased phosphorylation of the mitogen-activated protein kinases p38 and ERK1/2, whereas the specific inhibitors of p38, ERK1/2, and TTFA effectively blocked AGEs-induced reactive oxygen species production and eNOS downregulation.ConclusionsAGEs cause endothelial dysfunction by a mechanism associated with decreased eNOS expression and increased oxidative stress in HCAECs through activation of p38 and ERK1/2.

Tissue kallikrein-modified human endothelial progenitor cell implantation improves cardiac function via enhanced activation of akt and increased angiogenesis

Endothelial progenitor cells (EPCs) have been shown to enhance angiogenesis not only by incorporating into the vasculature but also by secreting cytokines, thereby serving as an ideal vehicle for gene transfer. As tissue kallikrein (TK) has pleiotropic effects in inhibiting apoptosis and oxidative stress, and promoting angiogenesis, we evaluated the salutary potential of kallikrein-modified human EPCs (hEPCs; Ad.hTK-hEPCs) after acute myocardial infarction (MI). We genetically modified hEPCs with a TK gene and evaluated cell survival, engraftment, revascularization, and functional improvement in a nude mouse left anterior descending ligation model. hEPCs were manipulated to overexpress the TK gene. In vitro, the antiapoptotic and paracrine effects were assessed under oxidative stress. TK protects hEPCs from oxidative stress-induced apoptosis via inhibition of activation of caspase-3 and -9, induction of Akt phosphorylation, and secretion of vascular endothelial growth factor. In vivo, the Ad.hTK-hEPCs were transplanted after MI via intracardiac injection. The surviving cells were tracked after transplantation using near-infrared optical imaging. Left ventricular (LV) function was evaluated by transthoracic echocardiography. Capillary density was quantified using immunohistochemical staining. Engrafted Ad.hTK-hEPCs exhibited advanced protection against ischemia by increasing LV ejection fraction. Compared with Ad.Null-hEPCs, transplantation with Ad.hTK-hEPCs significantly decreased cardiomyocyte apoptosis in association with increased retention of transplanted EPCs in the myocardium. Capillary density and arteriolar density in the infarct border zone was significantly higher in Ad.hTK-hEPC-transplanted mice than in Ad.Null-hEPC-treated mice. Transplanted hEPCs were clearly incorporated into CD31+ capillaries. These results indicate that implantation of kallikrein-modified EPCs in the heart provides advanced benefits in protection against ischemia-induced MI by enhanced angiogenesis and reducing apoptosis.

Biodistribution and Elimination Study of Fluorine-18 Labeled Nε-Carboxymethyl-Lysine following Intragastric and Intravenous Administration

Background Nε-carboxymethyl-lysine (CML) is a major advanced glycation end-product (AGEs) widely found in foods. The aim of our study was to evaluate how exogenous CML-peptide is dynamically absorbed from the gastrointestinal tract and eliminated by renal tubular secretion using microPET imaging. Methods The present study consisted of three investigations. In study I, we synthesized the imaging tracer 18F-CML by reacting N-succinimidyl 4-18F-fluorobenzoate (18F-SFB) with CML. In study II, the biological activity of 18F-CML was evaluated in RAW264.7 cells and HepG2 cells. In study III, the biodistribution and elimination of AGEs in ICR mice were studied in vivo following tail vein injection and intragastric administration of 18F-CML. Result The formation of 18F-CML was confirmed by comparing its retention time with the corresponding reference compound 19F-CML. The radiochemical purity (RCP) of 18F-CML was >95%, and it showed a stable character in vitro and in vivo. Uptake of 18F-CML by RAW264.7 cells and HepG2 cells could be inhibited by unmodified CML. 18F-CML was quickly distributed via the blood, and it was rapidly excreted through the kidneys 20 min after tail vein injection. However, 18F-CML was only slightly absorbed following intragastric administration. After administration of 18F-CML via a stomach tube, the radioactivity was completely localized in the stomach for the first 15 min. At 150 min post intragastric administration, intense accumulation of radioactivity in the intestines was still observed. Conclusions PET technology is a powerful tool for the in vivo analysis of the gastrointestinal absorption of orally administered drugs. 18F-CML is hardly absorbed by the gastrointestinal tract. It is rapidly distributed and eliminated from blood following intravenous administration. Thus, it may not be harmful to healthy bodies. Our study showed the feasibility of noninvasively imaging 18F-labeled AGEs and was the first to describe CML-peptide gastrointestinal absorption by means of PET.

Analysis of in situ and ex vivo αVβ3 integrin expression during experimental carotid atherogenesis.

Objective Mural inflammation has been shown to contribute to the development of plaque, with the αVβ3 integrin highly expressed in atherosclerotic plaques. We herein examined αVβ3 integrin expression as a function of carotid atherosclerosis formation in the apolipoprotein E-deficient (apoE−/−) mouse. Methods and results Constrictive collars were placed around the left common carotid arteries of apo E−/− mice maintained on a high-fat diet (n = 14). Before and 21 days following collar placement, in vivo serial magnetic resonance imaging (MRI) measurements of the carotid aortic diameter were performed using a 7T magnetic resonance (MR) scanner. Near- infrared fluorescence (NIRF) imaging was performed (n = 6) using an in vivo imaging system 0–24 hours following administration of 1.0 nmol c(RGDyK)-Cy5.5 via the tail vein. A competition experiment was performed by the co-injection of a saturating dose of bicyclic RGD peptide H-Glu[cyclo(Arg-Gly-Asp-D-Tyr-Lys)]2 (n = 3). Following image acquisition and sacrifice at 24 hours after injection, carotid arteries were harvested for histological analyses. Neointima formation and arterial remodeling in the carotid arteries of apoE−/− mice were induced by the placement of a constrictive collar. Significantly greater fluorescent signals were obtained from constrictive collar left common carotid arteries as compared to uninvolved aortic segments in constrictive collar mice. Binding to stenotic lesions was efficiently blocked in competition experiments. Immunostaining confirmed the presence of mural αVβ3 integrin expression in macrophages in the neointima. Signal intensity increased in a macrophage density-dependent fashion in the stenotic segments. Conclusion Mural αVβ3 integrin expression, as determined using RGD-Cy5.5 near-infrared optical imaging, was increased in carotid arteries with constrictive collars in experimental mice. This expression can estimate the macrophage-bound inflammatory activity of atherosclerotic lesions.