Cong Fu

Exposure to supernatants of macrophages that phagocytized dead mesenchymal stem cells improves hypoxic cardiomyocytes survival.

OBJECTIVE To observe the impact of supernatants from macrophages that phagocytized dead MSCs (pMΦ) on the survival of hypoxic cardiomyocytes. METHODS MSCs were isolated from bone marrow of mice and dead MSCs were harvested after 6h hypoxia. Macrophages were obtained from thioglycolate-elicited peritoneal cavity. Macrophages and dead MSCs were co-cultured for 2 days in the presence or absence of LPS (1 μg/ml). Cardiomyocytes obtained from neonatal mice were exposed to various medium including supernatants from pMΦ. MTT cell proliferation assay and mitochondria membrane potential were used to evaluate the viability of cardiomyocytes. Cytokines and chemokines (TNF-α, IFN-γ, IL-6, IL-12, PGE2, VEGF-α, Ang-1, KGF, IGF-1, PDGF-BB, and EPO) in culture medium of macrophages, MSCs and pMΦ were detected by ELISA and Real-Time-PCR. RESULTS phagocytic activity of macrophages to dMSC was significantly enhanced by LPS. PGE2, VEGF-α, Ang-1, KGF, IGF-1, PDGF-BB, and EPO levels were significantly increased in supernatants of pMΦ. Exposure to supernatants of pMΦ significantly improved viability and survival time of hypoxic cardiomyocytes. CONCLUSION Exposure to supernatants of pMΦ significantly improved viability and survival time of hypoxic cardiomyocytes, which might be linked to increased cytokines and chemokines secretion by pMΦ.

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.

Intermediate Monocytes Lead to Enhanced Myocardial Remodelling in STEMI Patients With Diabetes

This study aimed to evaluate the potential associations of intermediate monocytes (CD14(++)CD16(+)) with myocardial remodelling in ST segment elevation myocardial infarction (STEMI) patients with diabetes.A total of 67 STEMI patients with diabetes were enrolled. The control group consisted of 65 STEMI patients without diabetes. All patients received emergency medical services for reperfusion therapy in less than 12 hours after onset of AMI. Blinded to patient clinical characteristics, monocyte subset analysis was performed using flow cytometry immediately after admission. mRNA of Chemokine Decoy Receptor D6 in each subset of monocytes was validated by Q-PCR. Expression of CCL2 in patient plasma was determined with an Elisa kit. Infarct size and left ventricular ejection fraction (LVEF) were measured using 3-dimensional echocardiography 3 days and 6 months after AMI. The incidences of recurrent cardiovascular events and death in each group were measured using the Kaplan-Meier estimator in follow-up during the next 24 months. Cox proportional-hazard models were further used to analyze the relationship of monocyte cell counts and event-free survival after adjusting for confounding factors.The number of circulating intermediate monocytes was significantly correlated with LVEF% and infarct size (r = -0.32; P = 0.008; r = 0.57, P < 0.001) in STEMI patients with diabetes compared with those without diabetes 6 months after AMI. Chemokine Decoy Receptor D6 transcript levels were lower in intermediate monocytes of STEMI patients with diabetes compared to the subsets in STEMI patients without diabetes (P < 0.001). Higher levels of CCL2 (pg/mL) were observed in STEMI patients with diabetes compared to STEMI patients without diabetes (P < 0.001). During a mean follow-up period of 24 ± 1 month, recurrent cardiovascular events or death occurred in 23 patients belonging to the STEMI with diabetes group and 10 belonging to the control group. Univariate Kaplan-Meier analysis revealed that counts of the intermediate monocytes according to median showed statistical significance in STEMI patients with diabetes (P = 0.010). After full adjustment for confounding factors, the cells were found to remain independently related to recurrent cardiovascular events or death in this group (P = 0.004, 95% CI: 1.62-12.49).Intermediate monocytes were associated with LV remodelling in STEMI patients with diabetes. The cells were predictive for recurrent cardiovascular events or death in these patients. A low level of D6 mRNA in the intermediate monocytes of STEMI patients with diabetes and high level of CCL2 in these patients may partially explain the causality.

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.

Rationale and design of MANJUSRI trial: a randomized, open-label, active-controlled multicenter study to evaluate the safety of combined therapy with ticagrelor and warfarin in AF subjects after PCI-eS.

BACKGROUND Long-term oral anticoagulant treatment is obligatory in patients with atrial fibrillation (AF, score of CHA₂DS₂VASc≥2). When these patients undergo percutaneous coronary intervention with a drug-eluting stent (PCI-eS), there is also an indication for aspirin and clopidogrel treatment, according to the ESC Guidelines. However, triple therapy has been known to increase the risk of bleeding complications. Unfortunately, there is little prospective data available to resolve this issue. Therefore, it is imperative that an optimal therapy for AF patients with indications of both anticoagulation and antiplatelet intervention to prevent thrombotic complications without increasing the risk of bleeding is found. OBJECTIVES This prospective, randomized, multicenter study is going to assess the hypothesis that in persistent or permanent AF patients (score of CHA₂DS₂VASc≥2) after PCI-eS, the combination therapy of oral anticoagulation (warfarin) and ticagrelor (90 mg/bid) could reduce the risk of bleeding events. DESIGN A multicenter, active-controlled, open-label, randomized trial is to be performed to evaluate dual antithrombotic therapy (ticagrelor+warfarin) in persistent or permanent AF patients (score of CHA₂DS₂VASc≥2) after PCI-eS versus the combination of triple antithrombosis (clopidogrel+aspirin+warfarin). The primary endpoint is the overall bleeding up to 6 months, according to TIMI criteria and classifications. The secondary endpoints are the major bleeding events up to 6 months, according to TIMI criteria. The sample size is estimated at 296. CONCLUSION This study is intended to provide information about the safety characteristics of warfarin and ticagrelor in persistent or permanent AF patients after PCI-eS. No prospective randomized study has been conducted on the issue of antithrombotic therapy using warfarin and ticagrelor in these patients. Therefore, the MANJUSRI trial will help to explore and determine a new potential therapeutic regimen for AF patients after PCI-eS. TRIAL REGISTRATION Clinical Trials.gov # NCT02206815, registered July 30, 2014.

Pivotal Role of Regulator of G-protein Signaling 12 in Pathological Cardiac Hypertrophy.

Cardiac hypertrophy is a major predictor of heart failure and is regulated by diverse signaling pathways. As a typical multi-domain member of the regulator of G-protein signaling (RGS) family, RGS12 plays a regulatory role in various signaling pathways. However, the precise effect of RGS12 on cardiac hypertrophy remains largely unknown. In this study, we observed increased expression of RGS12 in the development of pathological cardiac hypertrophy and heart failure. We then generated genetically engineered mice and neonatal rat cardiomyocytes to investigate the effects of RGS12 during this pathological process. Four weeks after aortic banding, RGS12-deficient hearts showed decreased cardiomyocyte cross area (374.7±43.2 &mgr;m2 versus 487.1±47.9 &mgr;m2 in controls; P<0.05) with preserved fractional shortening (43.0±3.4% versus 28.4±2.2% in controls; P<0.05), whereas RGS12-overexpressing hearts exhibited increased cardiomyocyte cross area (582.4±46.7 &mgr;m2 versus 474.8±40.0 &mgr;m2 in controls; P<0.05) and reduced fractional shortening (20.8±4.1% versus 28.6±3.2% in controls; P<0.05). RGS12 also contributed to angiotensin II–induced hypertrophy in isolated cardiomyocytes. Mechanistically, our data indicated that the activation of MEK1/2–ERK1/2 signaling may be responsible for the prohypertrophic action of RGS12. In addition, the requirement of the MEK1/2–ERK1/2 signaling for RGS12-mediated cardiac hypertrophy was confirmed in rescue experiments using the MEK1/2-specific inhibitor U0126. In conclusion, our findings provide a novel diagnostic and therapeutic target for pathological cardiac hypertrophy and heart failure.

Correlation Between Serum Uric Acid and Renal Function in Patients With Stable Coronary Artery Disease and Type 2 Diabetes

Background The aim of this study was to investigate the relationship between serum uric acid and renal function, expressed as estimated glomerular filtration rate (eGFR), in patients with stable coronary artery disease (CAD) and type 2 diabetes mellitus (T2DM) in China. Methods Serum levels of uric acid and creatinine were determined in 526 enrolled patients diagnosed with stable CAD and T2DM. To assess renal function, eGFR was calculated using a modified MDRD formula suitable for the Chinese population. Patients’ anthropometric measurements were collected using standardized protocols, and 6-month follow-up results were collected and analyzed. Pearson’s correlation coefficient r was calculated and multivariate analysis was performed to evaluate the relationship between uric acid and renal function. Patients with eGFR < 60 mL/min/1.73 m2 were classified as having reduced renal function or chronic kidney disease (CKD) in this study. Results Uric acid levels were negatively associated with eGFR (P = 0.002), especially in patients with CKD (eGFR < 60 mL/min/1.73 m2) (P < 0.001). In patients with reduced renal function, the risk in the highest quartile of uric acid levels was higher than in the lowest quartile (odds ratio 9.18, 95% confidence interval: 4.01 - 21.01, P < 0.001). These associations remained after multiple adjustments for potential confounders and were recapitulated after 6-month follow-up. Conclusions Serum uric acid level is negatively associated with renal function, as assessed by eGFR, and serves as an independent predictor for CKD in patients with stable CAD and T2DM.

Bradykinin inhibits oxidative stress-induced senescence of endothelial progenitor cells through the B2R/AKT/RB and B2R/EGFR/RB signal pathways

Circulating endothelial progenitor cells (EPCs) have multiple protective effects that facilitate repair of damage to tissues and organs. However, while various stressors are known to impair EPC function, the mechanisms of oxidative stress-induced EPC senescence remains unknown. We demonstrated that B2 receptor (B2R) expression on circulating CD34+ cells was significantly reduced in patients with diabetes mellitus (DM) as compared to healthy controls. Furthermore, CD34+ cell B2R expression in patients with DM was inversely correlated with plasma myeloperoxidase concentrations. Bradykinin (BK) treatment decreased human EPC (hEPC) senescence and intracellular oxygen radical production, resulting in reduced retinoblastoma 1 (RB) RNA expression in H2O2-induced senescent hEPCs and a reversal of the B2R downregulation that is normally observed in senescent cells. Furthermore, BK treatment of H2O2-exposed cells leads to elevated phosphorylation of RB, AKT, and cyclin D1 compared with H2O2-treatment alone. Antagonists of B2R, PI3K, and EGFR signaling pathways and B2R siRNA blocked BK protective effects. In summary, this study demonstrates that BK significantly inhibits oxidative stress-induced hEPC senescence though B2R-mediated activation of PI3K and EGFR signaling pathways.

Tissue kallikrein is related to the severity of coronary artery disease

BACKGROUND The impairment of the tissue kallikrein (KLK1)-kinin system (KKS) may result in atheroma development. However, it remains unclear if the KKS correlates with coronary artery disease (CAD). METHODS KLK1, VEGF and hs-CRP plasma levels were measured in 100 patients newly diagnosed with CAD and 33 CAD-free controls. Patients were followed-up for the incidence of major adverse cardiovascular events (MACE) for 8months to 2y. Gene expression of KLK1, CD105 and CD68 was assessed in human coronary endarterectomy specimens. RESULTS Patients with CAD and acute coronary syndrome (ACS) had significantly elevated KLK1 levels. In addition, the concentration of hs-CRP was increased in ACS patients. A strong positive correlation between plasma KLK1 and the severity of CAD was also demonstrated, suggesting that high KLK1 levels are an independent predictor for CAD. MACE during follow-up significantly correlated with KLK1 levels in the ACS group. Unstable coronary plaques demonstrated markedly increased KLK1 levels, macrophage infiltration and high microvessel density. Additionally, KLK1 staining primarily colocalized with macrophages. CONCLUSIONS In the present study, plasma KLK1 levels were a useful predictor for the presence and extent of CAD. More extensive studies are, however, necessary in order to validate these findings.

Anti-connective tissue growth factor detects and reduces plaque inflammation in early-stage carotid atherosclerotic lesions

This study explored connective tissue growth factor (CTGF)-targeted ultrasmall superparamagnetic iron oxides (USPIOs) for noninvasive MRI of CTGF within carotid atherosclerotic lesions in apoE-deficient (apoE-/-) mice. Anti-CTGF polyclonal and nonspecific IgG antibodies were conjugated to polyethylene glycol-coated USPIOs, and apoE-/- carotid partial ligation-model mice were imaged via MRI before and after contrast administration. ApoE-/- mice were treated with CTGF-neutralizing antibodies for 3 weeks. Carotid artery diameter and plaque volume were measured via MRI in IgG and CTGF antibody-treated groups. Anti-CTGF-USPIO-treated macrophages showed the greatest iron uptake. MRI signal loss was observed in carotid atherosclerotic lesions 24 h after anti-CTGF-USPIO administration, consistent with the presence of nanoparticles, as indicated by pathological examinations. Atheromata in anti-CTGF-treated mice showed reduced macrophage deposition, CTGF expression, and plaque volume. Anti-CTGF-USPIOs can be used for the direct detection of CTGF and imaging of atherosclerotic lesions in vivo. CTGF is a potential therapeutic target for treating atherosclerosis.