Shuanglun Xie

Differentiated markers in undifferentiated cells: Expression of smooth muscle contractile proteins in multipotent bone marrow mesenchymal stem cells

In studying the differentiation of stem cells along smooth muscle lineage, smooth muscle cell (SMC) contractile proteins serve as markers for the relative state of maturation. Yet, recent evidence suggests that some SMC markers are probably expressed in multipotent mesenchymal stem cells (MSCs). Such a paradox necessitates investigations to re‐examine their role as differentiated markers in MSCs. We tried to detect the expression of four widely used SMC markers including α‐smooth muscle actin (α‐SMA), h1‐calponin, desmin and smooth muscle myosin heavy chain (SM‐MHC), as well as the other isoforms of calponin family in resting MSCs. Then we used three different conditions to initiate MSCs differentiation along SMC lineage, and examined the alternation of SMC markers expression at both the transcript level and protein level. Desmin and h1‐calponin are expressed in MSCs, in the presence or absence of SMC induction conditions. Moreover, MSCs are shown to express all known isoforms of calponin. Double‐staining reveals that h1‐calponin +/α‐SMA – cells constitute the majority of resting MSCs. Under differentiated conditions, expression of SM‐MHC was initiated and expression of α‐SMA was promoted. The expression of SM‐MHC and upregulation of α‐SMA are relatively reliable indications of a mature smooth muscle phenotype in MSCs. Given that the cells are particularly rich in calponins expression, we postulate possible roles of these proteins in regulating cellular function by taking part in actin cytoskeleton and signaling. These findings imply that an extensive study of the cell physiology of MSCs should focus on the functional roles for these proteins, rather than simply regard them as differentiated markers.

Inhibition of Protein Kinase C β2 Prevents Tumor Necrosis Factor-α-Induced Apoptosis and Oxidative Stress in Endothelial Cells: The Role of NADPH Oxidase Subunits

We investigate the cell signal transduction pathway protein kinase C (PKC) and the role of NADPH subunits in the process of TNF-α-induced endothelial apoptosis. Human umbilical vein endothelial cells (HUVEC) were treated with one of these: 1 mM PKC β2 inhibitor CGP53353, 10 mM PKC δ inhibitor rottlerin, combination CGP53353 with rottlerin, 3 ×10–4M NADPH oxidase inhibitor apocynin, 5 × 10–6M NADPH oxidase peptide inhibitor gp91ds-tat. The apoptosis process was assessed by Hoechst 33342 stain, flow cytometry and Western blot analysis, while intracellular reactive oxygen species (ROS) production was detected by 2,7’-dichlorodihydrofluorescein diacetate (DCFH-DA). The NADPH oxidase subunit gene and protein expression were assessed by quantitative real-time PCR and Western blot analysis, respectively. TNF-α significantly induced HUVEC apoptosis and ROS production, accompanying with dramatic upregulation of NADPH oxidase subunits: NOX2/gp91phox, NOX4, p47phox and p67phox, whereas these enhancements were abolished by the treatment with PKC inhibitors. High TNF-α level exposure induces HUVEC apoptosis, as well as a ROS generation increase via the PKC β2-dependent activation of NADPH oxidase. Although the PKC δ pathway may enhance TNF-α-induced HUVEC apoptosis, it does not involve the ROS pathway. Upregulation of expression of NADPH subunits is important in this process, which leads to a new target in antioxidative therapy for vascular disease prevention.

Optimal strategy of coronary revascularization in chronic kidney disease patients: a meta-analysis.

BACKGROUND Patients with chronic kidney disease (CKD) have high risks of coronary artery disease (CAD). Coronary revascularization is beneficial for long-term survival, but the optimal strategy remains still controversial. METHODS We searched studies that have compared percutaneous coronary intervention (PCI) and coronary artery bypass graft (CABG) for revascularization of the coronary arteries in CKD patients. Short-term (30 days or in-hospital) mortality, long-term (at least 12 months) all-cause mortality, cardiac mortality and the incidence of late myocardial infarction and recurrence of revascularization were estimated. RESULTS 28 studies with 38,740 patients were included. All were retrospective studies from 1977 to 2012. Meta-analysis showed that PCI group had lower short-term mortality (OR 0.55, 95% CI 0.41 to 0.73, P<0.01), but had higher long-term all-cause mortality (OR 1.29, 95% CI 1.23 to 1.35, P<0.01). Higher cardiac mortality (OR 1.08, 95% CI 1.01 to 1.15, P<0.05), higher incidence of late myocardial infarction (OR 1.78, 95% CI 1.65 to 1.91, P<0.01) and recurring revascularization rate (OR 2.94, 95%CI 2.15 to 4.01, P<0.01) is found amongst PCI treated patients compared to CABG group. CONCLUSIONS CKD patients with CAD received CABG had higher risk of short-term mortality but lower risks of long-term all-cause mortality, cardiac mortality and late myocardial infarction compared to PCI. This could be due to less probable repeated revascularization.

Role of Circulating Fibrocytes in Cardiac Fibrosis

Objective:It is revealed that circulating fibrocytes are elevated in patients/animals with cardiac fibrosis, and this review aims to provide an introduction to circulating fibrocytes and their role in cardiac fibrosis. Data Sources:This review is based on the data from 1994 to present obtained from PubMed. The search terms were “circulating fibrocytes” and “cardiac fibrosis”. Study Selection:Articles and critical reviews, which are related to circulating fibrocytes and cardiac fibrosis, were selected. Results:Circulating fibrocytes, which are derived from hematopoietic stem cells, represent a subset of peripheral blood mononuclear cells exhibiting mixed morphological and molecular characteristics of hematopoietic and mesenchymal cells (CD34+/CD45+/collagen I+). They can produce extracellular matrix and many cytokines. It is shown that circulating fibrocytes participate in many fibrotic diseases, including cardiac fibrosis. Evidence accumulated in recent years shows that aging individuals and patients with hypertension, heart failure, coronary heart disease, and atrial fibrillation have more circulating fibrocytes in peripheral blood and/or heart tissue, and this elevation of circulating fibrocytes is correlated with the degree of fibrosis in the hearts. Conclusions:Circulating fibrocytes are effector cells in cardiac fibrosis.

Inhibiting extracellular matrix metalloproteinase inducer maybe beneficial for diminishing the atherosclerotic plaque instability.

Atherosclerotic plaque rupture and local thrombosis activation in the artery cause acute serious incidents such as acute coronary syndrome and stroke. The exact mechanism of plaque rupture remains unclear but excessive degradation of the extracellular matrix scaffold by matrix-degrading metalloproteinases (MMPs) has been implicated as one of the major molecular mechanisms in this process. Convincing evidence is available to prove that extracellular matrix metalloproteinase inducer (EMMPRIN) induces MMP expression and is involved in the inflammatory responses in the artery wall. The inflammation and MMPs have been shown to play a critical role for atherosclerotic lesion development and progression. More recent data showed that increased EMMPRIN expression was associated with vulnerable atherosclerotic lesions. Therefore, we speculate that EMMPRIN may be pivotal for atherosclerotic plaque instability, and hence inhibition of EMMPRIN expression could be a promising approach for the prevention or treatment of atheroma instability.

Transcription factor decoys for activator protein-1 (AP-1) inhibit oxidative stress-induced proliferation and matrix metalloproteinases in rat cardiac fibroblasts

Activator protein-1 (AP-1), which is a transcription factor, is implicated in the transcriptional regulation of a wide range of genes that participate in cell proliferation and extracellular matrix production. This investigation was performed to test the hypothesis that transfection of cardiac fibroblasts (CFs) with sufficient amounts of decoy oligodeoxynucleotides (ODNs) containing the AP-1-binding site would result in binding to the transfactor AP-1, which would thereby prevent CF proliferation and matrix metalloproteinase (MMP) expression. CFs from Sprague-Dawley rat hearts were cultured and exposed to different concentrations of xanthine + xanthine oxidase (XXO) and AP-1 decoy ODNs. MMP expression was assayed after oxidative stress and transfection with AP-1 decoy ODNs by real-time quantitative polymerase chain reaction and Western blot. Cell growth was determined by the cell count. XXO significantly increased the DNA-binding activity of AP-1 in a dose-dependent manner. We found that transfection with AP-1 decoy ODNs strongly inhibited XXO-induced CF proliferation and MMP gene expression in vitro. Taken together, our data demonstrate that AP-1 is a key transcription factor that mediates CF proliferation and MMP synthesis under oxidative stress. Transfection with AP-1 decoy ODNs may be a novel strategy to inhibit CF proliferation and MMP synthesis.

Oxidized Low-Density Lipoprotein-Induced Cyclophilin A Secretion Requires ROCK-Dependent Diphosphorylation of Myosin Light Chain.

Objective: Accumulation of cyclophilin A (CyPA) within atherosclerotic lesions is thought to be implicated in the progression of atherosclerosis. However, the source of CyPA within atherosclerotic lesions is still unknown. The aim of this study is to determine the role of oxidized low-density lipoproteins (ox-LDL) in vascular smooth muscle cell (VSMC)-derived CyPA secretion and the underlying mechanism. Methods and Results: Abundant CyPA and α-smooth muscle actin (α-SMA) expressed in atherosclerotic lesions was observed in apolipoprotein E-deficient mice. ox-LDL induced CyPA secretion from a primary culture of rat aortic smooth muscle cells in a dose- and time-dependent manner. Sulfosuccinimidyloleate, a CD36 inhibitor, prevented the ox-LDL-induced CyPA secretion. Pre-exposure to either the actin-depolymerizing agent cytochalasin D or the actin-polymerizing agent jasplakinolide inhibited CyPA secretion induced by ox-LDL. Gene silencing of vesicle-associated membrane protein 2 suppressed ox-LDL-induced CyPA secretion. ox-LDL caused the phosphorylation of myosin light chain (MLC). Inhibition of MLC by blebbistatin reversed the secretion of CyPA and the phosphorylation of MLC induced by ox-LDL. MLC kinase inhibitor ML-7 reduced the monophosphorylation of MLC but did not reduce CyPA secretion. Pretreatment with the rho-associated coiled-coil kinase (ROCK) inhibitor Y27632 blocked diphosphorylation of MLC and secretion of CyPA induced by ox-LDL. Conclusions: ox-LDL-induced CyPA secretion requires vesicle transportation, actin remodeling and ROCK-dependent diphosphorylation of MLC. VSMC-derived CyPA induced by ox-LDL may be associated with increased CyPA expression in atherosclerotic lesions.

IL-33 promotes IL-10 production in macrophages: a role for IL-33 in macrophage foam cell formation

We evaluated the role of IL-10- in IL-33-mediated cholesterol reduction in macrophage-derived foam cells (MFCs) and the mechanism by which IL-33 upregulates IL-10. Serum IL-33 and IL-10 levels in coronary artery disease patients were measured. The effects of IL-33 on intra-MFC cholesterol level, IL-10, ABCA1 and CD36 expression, ERK 1/2, Sp1, STAT3 and STAT4 activation, and IL-10 promoter activity were determined. Core sequences were identified using bioinformatic analysis and site-specific mutagenesis. The serum IL-33 levels positively correlated with those of IL-10. IL-33 decreased cellular cholesterol level and upregulated IL-10 and ABCA1 but had no effect on CD36 expression. siRNA-IL-10 partially abolished cellular cholesterol reduction and ABCA1 elevation by IL-33 but did not reverse the decreased CD36 levels. IL-33 increased IL-10 mRNA production but had little effect on its stability. IL-33 induced ERK 1/2 phosphorylation and increased the luciferase expression driven by the IL-10 promoter, with the highest extent within the −2000 to −1752 bp segment of the 5′-flank of the transcription start site; these effects were counteracted by U0126. IL-33 activated Sp1, STAT3 and STAT4, but only the STAT3 binding site was predicted in the above segment. Site-directed mutagenesis of the predicted STAT3-binding sites (CTGCTTCCTGGCAGCAGAA→︀CTGCCTGGCAGCAGAA) reduced luciferase activity, and a STAT3 inhibitor blocked the regulatory effects of IL-33 on IL-10 expression. Chromatin immunoprecipitation (CHIP) confirmed the STAT3-binding sequences within the −1997 to −1700 and −1091 to −811 bp locus regions. IL-33 increased IL-10 expression in MFCs via activating ERK 1/2 and STAT3, which subsequently promoted IL-10 transcription and thus contributed to the beneficial effects of IL-33 on MFCs.

Effect of Metabolic Syndrome on Risk Stratification for Left Atrial or Left Atrial Appendage Thrombus Formation in Patients with Nonvalvular Atrial Fibrillation

Background: Metabolic syndrome (MS) is a risk factor for stroke and thromboembolism event. Left atrial or LA appendage (LA/LAA) thrombus is a surrogate of potential stroke. The relationship between MS and atrial thrombus remains unclear. In this study, we sought to investigate the effect of MS on risk stratification of LA/LAA thrombus formation in patients with nonvalvular atrial fibrillation (NVAF). Methods: This cross-sectional study enrolled 294 consecutive NVAF patients without prior anticoagulant and lipid-lowering therapies. LA/LAA thrombus was determined by transesophageal echocardiography. Risk assessment of LA/LAA thrombus was performed using the CHADS2, CHA2DS2-VASc, MS, CHADS2-MS, and CHA2DS2-VASc-MS scores. Logistic regression analyses were performed to determine which factors were significantly related to LA/LAA thrombus. Odds ratio (OR) including 95% confidence interval was also calculated. The predictive powers of different scores for the risk of LA/LAA thrombus were represented by C-statistics and compared by receiver operating characteristic (ROC) analysis. Results: LA/LAA thrombi were identified in 56 patients (19.0%). Logistic analysis showed that MS was the strongest risk factor for LA/LAA thrombus in NVAF patients (OR = 14.698, P < 0.001). ROC curve analyses revealed that the C-statistics of CHADS2-MS and CHA2DS2-VASc-MS was significantly higher than those of CHADS2 and CHA2DS2-VASc scores (CHADS2-MS vs. CHADS2, 0.807 vs. 0.726, P = 0.0019). Furthermore, MS was helpful for identifying individuals with a high risk of LA/LAA thrombus in the population with a low risk of stroke (CHADS2 or CHA2DS2-VASc score = 0). Conclusions: MS is associated with LA/LAA thrombus risk in patients with NVAF. In addition to the CHADS2 and CHA2DS2-VASc scores, the CHADS2-MS and CHA2DS2-VASc-MS scores provide additional information on stroke risk assessment.

Interleukin 18 and extracellular matrix metalloproteinase inducer cross-regulation: implications in acute myocardial infarction.

Circulating interleukin-18 (IL-18) is thought to promote atherosclerosis and cardiovascular complications such as plaque rupture. Atherosclerosis is also characterized by smooth muscle cell migration, a consequence of extracellular matrix (ECM) degradation regulated by metalloproteinases (MMPs). Because extracellular matrix metalloproteinase inducer (EMMPRIN) has been shown to promote plaque instability by inducing ECM degradation and MMP synthesis, we investigated whether a cross-regulatory interaction exists between IL-18 and EMMPRIN in human monocytes. EMMPRIN levels in monocytes were markedly greater in 20 patients with acute myocardial infarction (AMI) compared with 20 patients with stable angina pectoris or 20 healthy volunteers (control group). The levels of IL-18 and MMP-9 in serum were also significantly greater in the AMI group in comparison with the other 2 groups. IL-18 levels positively correlated with increased levels of EMMPRIN in monocytes. In vitro, the expression of EMMPRIN was increased in monocytes cultured with IL-18, and IL-18 secretion was augmented in monocytes cultured with EMMPRIN. Gene silencing of EMMPRIN by small interfering RNA reduced monocyte secretion of both IL-18 and MMP-9. In the present study, cross-regulation between IL-18 and EMMPRIN in monocytes was demonstrated. This interaction may amplify the inflammatory cascade and be responsible for increased monocytic MMP-9 serum levels in atherosclerosis, contributing to atherosclerotic plaque destabilization and subsequent AMI.