Rui-Xia Xu

Lysophosphatidic acid protects mesenchymal stem cells against hypoxia and serum deprivation-induced apoptosis.

Bone marrow‐derived mesenchymal stem cells (MSCs) have shown great promise for cardiac repair. However, poor viability of transplanted MSCs within the ischemic heart has limited their therapeutic potential. Our previous studies have documented that hypoxia and serum deprivation (hypoxia/SD), induced MSCs apoptosis through the mitochondrial apoptotic pathway. Since serum lysophosphatidic acid (LPA) levels are known to be significantly elevated after acute myocardial infarction and that LPA enhanced survival of other cell systems, we embarked on determining whether LPA protects MSCs against hypoxia/SD‐induced apoptosis. We have also investigated the potential mechanism(s) that may mediate such actions of LPA. All experiments were carried out on rat bone marrow MSCs. Apoptosis was induced by exposure of cells to hypoxia/SD in a sealed GENbox hypoxic chamber. Effects of LPA were investigated in the absence and presence of inhibitors that target either Giproteins, the mitogen activated protein kinases ERK1/2, or phosphoinositide 3‐kinase (PI3K). The data obtained showed that hypoxia/SD‐induced apoptosis was significantly attenuated by LPA through Gi‐coupled LPA1 receptors linked to the downstream ERK1/2 and PI3K/Akt signaling pathways that function in parallel. Additional studies have demonstrated that hypoxia/SD‐induced activation of mitochondrial dysfunction was virtually abolished by LPA treatment and that inhibition of the LPA1 receptor, Gi proteins, the PI3K/Akt pathway, or ERKs effectively reversed this protective action of LPA. Taken together, our findings indicate that LPA is a novel, potent survival factor for MSCs and this may prove to be of considerable therapeutic significance in terms of exploiting MSC‐based therapy in the infracted myocardium.

Lovastatin protects mesenchymal stem cells against hypoxia- and serum deprivation-induced apoptosis by activation of PI3K/Akt and ERK1/2.

Cell therapy with bone marrow‐derived mesenchymal stem cells (MSCs) has been shown to have great promises in cardiac repair after myocardial infarction. However, poor viability of transplanted MSCs in the infracted heart has limited the therapeutic efficacy. Our previous studies have shown in vitro that rat MSCs undergo caspase‐dependent apoptosis in response to hypoxia and serum deprivation (Hypoxia/SD). Recent findings have implicated statins, an established class of cholesterol‐lowering drugs, enhance the survival of cells under various conditions. In this study, we investigated the effect of lovastatin on rat MSCs apoptosis induced by Hypoxia/SD, focusing in particular on regulation of mitochondrial apoptotic pathway and the survival signaling pathways. We demonstrated that lovastatin (0.01–1 µM) remarkably prevented MSCs from Hypoxia/SD‐induced apoptosis through inhibition of the mitochondrial apoptotic pathway, leading to attenuation of caspase‐3 activation. The loss of mitochondrial membrane potential and cytochrome‐c release from mitochondria to cytosol were significantly inhibited by lovastatin. Furthermore, the antiapoptotic effect of lovastatin on mitochondrial apoptotic pathway was effectively abrogated by both PI3K inhibitor, LY294002 and ERK1/2 inhibitor, U0126. The phosphorylations of Akt/GSK3β and ERK1/2 stimulated by lovastatin were detected. The activation of ERK1/2 was inhibited by a PI3K inhibitor, LY294002, but U0126, a ERK1/2 inhibitor did not inhibit phosphorylation of Akt and GSK3β. These data demonstrate that lovastatin protects MSCs from Hypoxia/SD‐induced apoptosis via PI3K/Akt and MEK/ERK1/2 pathways, suggesting that it may prove a useful therapeutic adjunct for transplanting MSCs into damaged heart after myocardial infarction. J. Cell. Biochem. 103: 256–269, 2008.

Association of plasma PCSK9 levels with white blood cell count and its subsets in patients with stable coronary artery disease

OBJECTIVE Recent studies have suggested that proprotein convertase subtilisin/kexin type 9 (PCSK9) is associated with atherosclerosis and plays a potential role in inflammation. However, the correlation between PCSK9 and white blood cell count (WBCC) has not yet been assessed. The objective of the present study was to examine the association of the WBCC and its subset counts with plasma PCSK9 levels in patients with stable coronary artery disease (CAD). METHODS In this cross-sectional study, a total of 251 consecutive, stable CAD patients who were not treated with lipid-lowering drugs were enrolled at our center between October 2012 and October 2013. The baseline clinical characteristics were collected, and the plasma PCSK9 levels were determined using ELISA. The associations of plasma PCSK9 levels with the WBCC and its subsets were investigated. RESULTS In the overall population, plasma PCSK9 levels were positively associated with the WBCC (r = 0.167, p = 0.008). Multivariable regression analysis revealed that the plasma PCSK9 levels were significantly and independently associated with the WBCC (β = 0.217, p < 0.001) and its subsets (neutrophil β = 0.152, p < 0.05; lymphocyte β = 0.241, p < 0.001). However, the relationships between PCSK9 and WBCC and its subsets remained significant in men (WBCC r = 0.234, p = 0.001; neutrophil r = 0.181, p = 0.014; lymphocyte r = 0.226, p = 0.002) but were not significant in women when the analysis was performed based on gender. CONCLUSIONS These data demonstrate that the plasma PCSK9 levels are independently associated with the WBCC and its subsets, suggesting a potential interaction between PCSK9 and chronic inflammation in patients with CAD.

Mesenchymal stem cells promote cardiomyocyte hypertrophy in vitro through hypoxia-induced paracrine mechanisms.

1 Mesenchymal stem cell (MSC) therapy for myocardial infarction has received increased attention since transplanted MSC were shown to improve cardiac function by transdifferentiating into cardiomyocytes and endothelial cells. However, recent studies have demonstrated that other mechanisms may contribute to the improvement in cardiac function observed after transplantation of MSC. The paracrine effect of MSC on cardiomyocyte is not clear. Thus, in the present study, we investigated the paracrine effect of MSC on the growth of neonatal rat cardiomyocytes in vitro. 2 Samples of MSC‐conditioned medium (MSC‐CM) were collected after rat MSC had been cultured under conditions of hypoxia and serum deprivation for 0, 3, 6, 9 or 24 h. Cardiomyocytes were then stimulated with the MSC‐CM for 48 h. Then, the protein content, cell area, [3H]‐leucine incorporation and atrial natriuretic factor‐luciferase (ANF‐Luc) expression of cardiomyocytes were measured. 3 The data showed that MSC‐CM collected at different time points had different effects and that MSC‐CM collected at 6 h significantly promoted cardiomyocyte hypertrophy by increasing total protein content, cell area, [3H]‐leucine incorporation and ANF gene expression. 4 In conclusion, MSC‐CM promoted cardiomyocyte hypertrophy in a paracrine manner. The results provide a better understanding of the mechanisms underlying the improvement in heart function after MSC transplantation.

Proprotein convertase subtilisin-kexin type 9 as a biomarker for the severity of coronary artery disease

Abstract Aim. To evaluate the relation of proprotein convertase subtilisin-kexin type 9 (PCSK9) levels to coronary artery disease (CAD). Methods. A total of 1031 consecutive individuals (552 CAD and 479 controls) were prospectively enrolled. The associations of plasma PCSK9 levels with the incidence and severity of CAD were investigated. Further, mediator analysis was performed to detect the potential mechanisms of the associations. Results. No difference in PCSK9 levels between CAD patients and controls was detected (median 224.75 versus 224.64 ng/mL, P > 0.05). However, the CAD group had higher PCSK9 levels than the control group when adjusting for the confounding factors (228.03 ± 1.01 versus 219.28 ± 1.02 ng/mL, P = 0.019). PCSK9 levels were also associated with the severity of CAD assessed by the Gensini score (GS) system (P for trend < 0.05). Logistic regression analysis showed that PCSK9 levels were associated with an increased CAD risk (OR 3.296 and 5.130 for the incidence and severity, respectively). Importantly, mediator analysis indicated that the effects of PCSK9 levels on CAD were mediated by lipid (around 20%) and inflammation (around 15%). Conclusions. PCSK9 levels were positively associated with the severity of CAD; the relatively important mechanisms including lipid and inflammation pathways were partly involved in this association.

Protective effects of icariin on cognitive deficits induced by chronic cerebral hypoperfusion in rats.

1 Icariin is a major constituent of flavonoids derived from the Chinese medicinal herb Epimedium revicornum Maxim. The aim of the present study was to investigate whether icariin has protective effects on learning ability and memory in a rat model of chronic cerebral hypoperfusion. 2 Chronic cerebral hypoperfusion was induced by permanent ligation of the common carotid artery in Wistar rats for 4 months. One month after permanent artery occlusion, rats were adminitered icariin at doses of 0, 30, 60 or 120 mg/kg per day, p.o., for 3 months. Neurobehavioural and neurobiochemical parameters were examined to evaluate the effects of icariin on cognitive deficits induced by chronic cerebral hypoperfusion. 3 The Morris water maze test revealed that learning ability and memory were severely impaired in untreated rats, but were significantly improved in icariin‐treated rats. Icariin treatment also ameliorated chronic cerebral hypoperfusion‐induced oxidative stress in the brain, as evidenced by reduced malondialdehyde formation and maintained superoxide dismutase activity. In addition, the decreased hippocampal levels of acetylcholine, acetylcholinesterase and choline acetyltransferase associated with chronic cerebral hypoperfusion were significantly prevented by icariin treatment. 4 In conclusion, icariin protects against cognitive deficits induced by chronic cerebral hypoperfusion in rats. These effects appear to be mediated through its anti‐oxidant effects, as well as its effects on the circulatory and cholinergic systems.

Specific LPA receptor subtype mediation of LPA-induced hypertrophy of cardiac myocytes and involvement of Akt and NFκB signal pathways

Lysophosphatidic acid (LPA) is a bioactive phospholipid with diverse functions mediated via G‐protein‐coupled receptors (GPCRs). In view of the elevated levels of LPA in acute myocardial infarction (MI) patients we have conducted studies aimed at identifying specific LPA receptor subtypes and signaling events that may mediate its actions in hypertrophic remodeling. Experiments were carried out in cultured neonatal rat cardiomyocytes (NRCMs) exposed to LPA and in a rat MI model. In NRCMs, LPA‐induced hypertrophic growth was completely abrogated by DGPP, an LPA1/LPA3 antagonist. The LPA3 agonist OMPT, but not the LPA2 agonist dodecylphosphate, promoted hypertrophy as examined by 3[H]‐Leucine incorporation, ANF‐luciferase expression and cell area. In in vivo experiments, LPA1, LPA2 and LPA3 mRNA levels as well as LPA1 and LPA3 protein levels increased together with left ventricular remodeling (LVRM) after MI. In addition, LPA stimulated the phosphorylation of Akt and p65 protein and activated NF‐κB‐luciferase expression. Inhibitors of PI3K (wortmannin), mTOR (rapamycin), and NF‐κB (PDTC or SN50) effectively prevented LPA‐induced 3[H]‐Leucine incorporation and ANF‐luciferase expression. Furthermore, ERK inhibitors (U0126 and PD98059) suppressed LPA‐stimulated activation of NF‐κB and p65 phosphorylation whereas wortmannin showed no effect on NF‐κB activation. Our findings indicate that LPA3 and/or LPA1 mediate LPA‐induced hypertrophy of NRCMs and that LPA1 and LPA3 may be involved in LVRM of MI rats. Moreover, Akt and NF‐κB signaling pathways independently implicate in LPA‐stimulated myocardial hypertrophic growth. J. Cell. Biochem. 103: 1718–1731, 2008.

Metformin and Resveratrol Inhibited High Glucose-Induced Metabolic Memory of Endothelial Senescence through SIRT1/p300/p53/p21 Pathway

Endothelial senescence plays crucial roles in diabetic vascular complication. Recent evidence indicated that transient hyperglycaemia could potentiate persistent diabetic vascular complications, a phenomenon known as “metabolic memory.” Although SIRT1 has been demonstrated to mediate high glucose-induced endothelial senescence, whether and how “metabolic memory” would affect endothelial senescence through SIRT1 signaling remains largely unknown. In this study, we investigated the involvement of SIRT1 axis as well as the protective effects of resveratrol (RSV) and metformin (MET), two potent SIRT1 activators, during the occurrence of “metabolic memory” of cellular senescence (senescent “memory”). Human umbilical vascular endothelial cells (HUVECs) were cultured in either normal glucose (NG)/high glucose (HG) media for 6 days, or 3 days of HG followed by 3 days of NG (HN), with or without RSV or MET treatment. It was shown that HN incubation triggered persistent downregulation of deacetylase SIRT1 and upregulation of acetyltransferase p300, leading to sustained hyperacetylation (at K382) and activation of p53, and subsequent p53/p21-mediated senescent “memory.” In contrast, senescent “memory” was abrogated by overexpression of SIRT1 or knockdown of p300. Interestingly, we found that SIRT1 and p300 could regulate each other in response to HN stimulation, suggesting that a delicate balance between acetyltransferases and deacetylases may be particularly important for sustained acetylation and activation of non-histone proteins (such as p53), and eventually the occurrence of “metabolic memory.” Furthermore, we found that RSV or MET treatment prevented senescent “memory” by modulating SIRT1/p300/p53/p21 pathway. Notably, early and continuous treatment of MET, but not RSV, was particularly important for preventing senescent “memory.” In conclusion, short-term high glucose stimulation could induce sustained endothelial senescence via SIRT1/p300/p53/p21 pathway. RVS or MET treatment could enhance SIRT1-mediated signaling and thus protect against senescent “memory” independent of their glucose lowering mechanisms. Therefore, they may serve as promising therapeutic drugs against the development of “metabolic memory.”

Proprotein Convertase Subtilisin/kexin type 9, C-reactive Protein, Coronary Severity, and Outcomes in Patients With Stable Coronary Artery Disease: A Prospective Observational Cohort Study

AbstractProprotein convertase subtilisin/kexin type 9 (PCSK9) is suggested as a novel factor associated with coronary artery disease (CAD). However, few studies have comprehensively evaluated plasma PCSK9 with cardiovascular risk till now. Hence, we aimed to prospectively investigate the association between baseline PCSK9 and cardiovascular risk graded with number of risk factors (RFs), coronary severity, and outcomes in patients with stable CAD.Baseline characteristics and biomarkers were measured in 616 consecutive, nontreated patients with stable CAD. Coronary severity was measured using SYNTAX, Gensini, and Jeopardy scoring systems. Patients were then received treatment and followed for a median of 17 months. The primary endpoints were cardiac death, stroke, myocardial infarction (MI), post-discharge revascularization, or unstable angina (UA).Overall, follow-up data were obtained from 603 patients. A total of 72 (11.9%) patients presented with at least 1 major adverse cardiovascular event (MACE) (4 cardiac deaths, 4 strokes, 6 MIs, 28 revascularizations, and 30 UAs). At baseline, PCSK9 was increased with an increasing number of RFs and positively associated with coronary severity scores (P < 0.05, all). After follow-up, those with MACE had a higher baseline PCSK9, hs-CRP, and coronary scores than those without (P < 0.05, all). Multivariate analysis showed that PCSK9, hs-CRP, and coronary scores were independently predictive for MACEs (P < 0.05, all). Interestingly, more significant predictive values of PCSK9 in medical-alone-treated population but no such associations in revascularization-treated patients were found.Together, plasma PCSK9, as well as hs-CRP and coronary scores, could independently predict MACEs in patients with stable CAD.

High-density lipoprotein subfractions in relation with the severity of coronary artery disease: A Gensini score assessment.

BACKGROUND The exact role of different high-density lipoprotein (HDL) subfractions in the pathogenesis of coronary artery disease (CAD) has not yet been fully explored. The aim of the present study was to examine the relationship between HDL subfractions and the severity of CAD in patients without statin therapy. METHODS A total of 382 consecutive patients (mean: 55.36 ± 11.30 years of age) who underwent coronary angiography from angina-like chest pain were investigated. Patients were classified into 2 groups according to the angiographic results: a CAD group (n = 283) and a control group (n = 99). The distribution of HDL subfractions was analyzed using a Quantimetrix Lipoprint HDL system. CAD severity was measured by Gensini score (GS). RESULTS Compared with the control group, HDL-cholesterol (HDL-C), large HDL-C level, and the large HDL subfraction percentages in the CAD group were significantly lower (P = .002, P < .001, P < .001, respectively). Meanwhile, a small HDL-C level and the percentage of small HDL subfraction were significantly higher (P = .003, P < .001, respectively). Correlation analysis showed that the percentage of a large HDL subfraction was negatively correlated with GS (β = -0.191, P = .005), whereas the percentage of a small HDL subfraction positively correlated with GS (β = 0.145, P = .023) in patients with CAD. CONCLUSIONS Small HDL subfraction was associated with the presence of CAD, whereas the percentage of large HDL and small HDL subfraction was negatively and positively associated with the severity of CAD, respectively.