Overexpression of PDZ1 domain prevents apoptosis of rat hippocampal neurons induced by kainic acid.

In our previous studies, Tat-GluR6-9c (a glutamate receptor 6 C-terminus peptide fused the TAT protein transduction sequence) not only inhibited the activation of MLK3 (mixed lineage kinase 3) and JNK (c-Jun N-terminal kinase) via the GluR6.PSD-95 (postsynaptic density protein 95).MLK3 signaling module but also diminished neuronal death induced by kainic acid or transient cerebral ischemia in rat hippocampus. Here, we investigate whether overexpression of the PDZ1 domain of PSD-95 protein could suppress the binding of GluR6 with PSD-95 and the activation of MLK3, MKK7 (mitogen-activated kinase kinase 7) and JNK1/2, and rescused neuronal cell death induced by kainic acid. Our results showed that overexpression of the PDZ1 domain of PSD-95 protein could prevent nuclear accumulation and abrogate neuronal cell death in SD (Sprague-Dawley) rat hippocampal neuronal cells. Further studies indicated that overexpression of PDZ1 could inhibit the enhancement of binding of GluR6 to PSD-95 and prevent the activation of MLK3, MKK7 and JNK1/2 induced by kainic acid. Taken together, the essential role of the PDZ1 domain of PSD-95 in apoptotic cell death in neurons provides an experimental foundation for gene therapy of neurodegenerative diseases with overexpression of the PDZ1 domain.

Enhanced cardiac sympathetic afferent reflex involved in sympathetic overactivity in renovascular hypertensive rats.

Sympathetic outflow is increased in hypertension. The aim of the present study was to investigate whether the cardiac sympathetic afferent reflex (CSAR) is enhanced in two-kidney one-clip (2K1C) renovascular hypertensive rats, and whether the enhanced CSAR contributes, in part, to the increased sympathetic outflow. Furthermore, the role of central angiotensin II type 1 (AT(1)) receptors in mediating the CSAR was determined. Under urethane and alpha-chloralose anaesthesia, the renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) were recorded in sinoaortic denervated and cervical vagotomized rats. The CSAR was evaluated by the response of RSNA and MAP to epicardial application of 1.0 nmol of capsaicin. Compared with sham-operated rats, the CSAR, baseline RSNA and plasma noradrenaline level were significantly enhanced in 2K1C rats. Intrapericardial administration of resiniferatoxin, which abolishes the CSAR because of the desensitization of transient receptor potential vanilloid 1-containing cardiac afferent fibres, decreased the RSNA and MAP. The enhanced CSAR in 2K1C rats was normalized by intracerebroventricular administration of the AT(1) receptor antagonist losartan. Intracerebroventricular administration of angiotensin II further potentiated the enhanced CSAR in 2K1C rats, a response which was abolished by pretreatment with losartan. These results indicate that the CSAR is enhanced in 2K1C rats and the enhanced CSAR contributes, in part, to the sympathetic activation and hypertension. Central AT(1) receptors are involved in the enhanced CSAR in 2K1C rats.

Targeted correction of point mutations in the low density lipoprotein receptor gene mediated by single-stranded oligonucleotides in vivo.

This study was designed to investigate the repair of point mutations in the low density lipoprotein receptor (LDLR) gene mediated by single-stranded oligonucleotides (SSOs) in vivo. Mutations in the LDLR gene are known to be the prime cause of familial hypercholesterolemia (FH). SSOs result in sequence-specific alterations leading to the correction of mutations. In the present study, the LDLR gene with a nonsense mutation (c660x) was fused to a luciferase reporter gene (p660-LDLR-luc) and introduced into mouse liver by hydrodynamic gene transfer. These mice were then injected via the tail vein with different SSOs complexed with polyethylenimine. Firefly luciferase activity present in hepatic cell lysate was measured to analyze repair efficiency. Restriction fragment length polymorphism analysis and direct sequencing were performed to affirm that the LDLR mutation was corrected. The results indicate that the LDLR mutation was corrected in the liver in vivo only in the presence of antisense SSOs (anti-SSOs). Our findings provide initial evidence that the point mutation in p660-LDLR-luc can be corrected by anti-SSO targeted repair in vivo. This may be a potential strategy for the treatment of FH.

A genetic variant of apolipoprotein M increases susceptibility to coronary artery disease in a Chinese population.

1. High-density lipoprotein (HDL) is widely accepted as a lipoprotein that protects against coronary artery and other atherosclerotic diseases. Recently, a new apolipoprotein encoded by the APOM gene, which plays an important role in affecting the intrinsic properties of HDL, has been reported. Genetic variations exist in the APOM gene, but their significance is presently unclear. The aim of the present study was to elucidate whether the APOM T-855C mutant allele is implicated in coronary artery disease (CAD). 2. In the present study, 418 patients with CAD and 372 controls were studied, all of whom were Han Chinese from Jiangsu Province, China. Plasma levels of triglycerides (TG), total cholesterol (TC), HDL-cholesterol (HDL-C) and low-density lipoprotein-cholesterol (LDL-C) were evaluated. Genomic DNA from the whole blood from these subjects was subjected to polymerase chain reaction amplification and restriction enzyme digestion to determine genotype with respect to the APOM T-855C polymorphism. 3. The allelic frequencies were in Hardy-Weinberg equilibrium. Plasma HDL levels were significantly lower in subjects with CAD than in control subjects (1.08 +/- 0.31 vs 1.25 +/- 0.32, respectively; P < 0.001) and the distribution of genotypes and allelic frequencies was significantly different in the two groups (P = 0.013 and 0.005, respectively). Multiple logistic regression analysis after adjustment for age, gender, smoking, body mass index, hypertension and serum glucose showed that, compared with the wild-type TT genotype, carriers of the C allele had an increased risk of CAD (odds ratio = 1.819, 95% confidence interval 1.142-2.898; P = 0.012). 4. In conclusion, the results of the present study suggest that the APOM T-855C polymorphism carries an increased risk for CAD in this Chinese population.

Abnormal surface markers expression on bone marrow CD34+ cells and correlation with disease activity in patients with systemic lupus erythematosus.

Defects of hematopoietic stem cells (HSCs) have been suggested to contribute to the development of systemic lupus erythematosus (SLE). The aim of this study was to investigate the phenotypic characteristics of bone marrow (BM) CD34(+) cells in patients with SLE and its relationship with SLE disease activity. Ten SLE patients and 10 healthy subjects were recruited and their BM CD34(+) cells were analyzed by flow cytometric analysis with CD45/SSC gating for the expression of CD90, CD95, CD117, CD123, CD164, CD166, FAS-L, and HLA-DR. The percentage of BM CD34(+) cells was significantly decreased in active SLE patients (1.48 +/- 0.41%, n = 7) compared to the healthy controls (2.31 +/- 0.75%, n = 10, p < 0.01), but no significant difference was found between the inactive patients (2.04 +/- 0.44%, n = 3) and the controls. The expression of CD95, CD123, and CD166 on BM CD34(+) cells were significantly increased in SLE patients (48.31 +/- 10.59%, 44.9 +/- 21.5%, 30.9 +/- 19.54%, respectively, n = 10) when compared with the control subjects (24.33 +/- 11.1%, 19.5 +/- 4.4%, 10.7 +/- 5.5%, respectively, n = 10, p < 0.05). The increased CD123 expression was negatively correlated with the number of peripheral white blood cells (r = -0.700, p < 0.05, n = 10). The percentage of CD166 expression was found significantly correlated with the index of SLE disease activity (r = 0.472, p < 0.05, n = 10) and 24 h proteinuria (r = 0.558, p < 0.05, n = 10), but negatively correlated with serum C3 level (r = -0.712, p < 0.01, n = 10). Our study found that the surface marker expression of CD95, CD123, and CD166 on BM CD34(+) cells were significantly increased in patients. This supports the hypothesis that there are abnormalities of the HSC in SLE. Since CD166 and CD123 correlated with the overall lupus activity, their role as a biomarker of inflammatory disease activity also requires further study.

Ultrasound-targeted microbubble destruction improves the low density lipoprotein receptor gene expression in HepG2 cells.

Ultrasound-targeted microbubble destruction had been employed in gene delivery and promised great potential. Liver has unique features that make it attractive for gene therapy. However, it poses formidable obstacles to hepatocyte-specific gene delivery. This study was designed to test the efficiency of therapeutic gene transfer and expression mediated by ultrasound/microbubble strategy in HepG2 cell line. Air-filled albumin microbubbles were prepared and mixed with plasmid DNA encoding low density lipoprotein receptor (LDLR) and green fluorescent protein. The mixture of the DNA and microbubbles was administer to cultured HepG2 cells under variable ultrasound conditions. Transfection rate of the transferred gene and cell viability were assessed by FACS analysis, confocal laser scanning microscopy, Western blot analysis and Trypan blue staining. The result demonstrated that microbubbles with ultrasound irradiation can significantly elevate exogenous LDLR gene expression and the expressed LDLRs were functional and active to uptake their ligands. We conclude that ultrasound-targeted microbubble destruction has the potential to promote safe and efficient LDLR gene transfer into hepatocytes. With further refinement, it may represent an effective nonviral avenue of gene therapy for liver-involved genetic diseases.

[Study on the association of ABCA1 gene common variants with the risk of coronary atherosclerotic heart disease].

OBJECTIVE: To investigate the association of R219K and M883I polymorphisms of ATP binding cassette transporter 1 gene with lipid metabolism and the susceptibility to coronary atherosclerotic heart disease in Chinese population. METHODS: Genotypes were determined by PCR-restriction fragment length polymorphism and Primer introduced restriction analysis-PCR techniques, respectively, in 248 unrelated CHD-free controls and 224 CHD cases. RESULTS: Smoking, high blood pressure and high serum glucose were independent risk factors for CHD. Multivariate logistic regression analysis revealed that individuals carrying at least one 219K variant allele (RK + KK genotypes) had a significantly decreased risk for CHD (adjusted OR = 0.41; 95% CI = 0.27-0.61) compared with the wild-type genotype (219RR) and only 883II homozygotes displayed a decreased risk for CHD (adjusted OR = 0.54; 95% CI = 0.26-1.11) compared with 883MM and 883MI genotypes. Furthermore, compared with individuals with both wild genotypes (219 RR and 883 MM or 883 MI) other individuals with all other assembly genotypes had a significantly decreased risk (adjusted OR = 0.39, 95% CI = 0.26-0.60). Plasma HDL-C in 219K allele carriers were markedly higher than those in 219 K non-carriers in controls (P = 0.037). CONCLUSION: The ABCA1 R219K polymorphism may be involved in the variability of serum HDL-C and the susceptibility to coronary artery disease.

Association between apolipoprotein A5 - 1131T > C polymorphism and susceptibility of coronary artery disease in Chinese.

OBJECTIVE: To investigate the relationship between apolipoprotein A5(apoA5) - 1131T > C polymorphism and the susceptibility of coronary artery disease (CAD) in Chinese. METHODS: The restriction fragment length polymorphism of apoA5 gene - 1131T > C was studied using PCR in a case-control study which enrolled 235 patients with CAD diagnosed by angiography and 262 healthy controls from Jiangsu province. RESULTS: The frequencies of T, C allele were 59.57%ì40.43% and 65.65%, 34.35% in CAD group and control group respectively. There was statistically significant difference in allele frequencies between CAD group and control group (P < 0.05). The susceptibility to CAD for the CC genotype was much higher than that for wild type TT (OR = 1.872, 95% CI = 1.039 - 3.376, P = 0.037), even after the use of Logistic regression models (OR = 2.285, 95% CI = 1.222 - 4.274, P = 0.012). In control group, there was significant difference in TG levels among different genotypes, the C allele carriers had higher serum TG concentration (P = 0.007). CONCLUSION: apoA5 - 1131T > C polymorphism is associated with an increased risk of CAD and is also in strong association with serum TG levels.

Ultrasound/microbubble enhances foreign gene expression in ECV304 cells and murine myocardium.

Although viral vectors are efficient systems to transfer foreign genes into cells or target tissues, safety issues remain in relation to human gene therapy. Microbubbles currently used as ultrasound contrast agents have been applied in transfection of genes. This study was designed to test the transfection efficiency and the expression of exogenous gene mediated by ultrasound irradiation enhanced air filled albumin microbubbles in ECV304 cell line in vitro and the heart of the mouse in vivo. Air filled microbubbles (2.0-4.0 microm in diameter) were created by sonicating the mixture of human albumin, glucose, mannitol and special additive that was designed for stabilization. Plasmid DNA loading the reporter genes was gently mixed with microbubbles. The mixture of plasmid DNA and microbubbles was administrated to cultured ECV304 cells and BALB/c mice (tail vein injection) under different ultrasound/microbubble conditions, and then the transfection and expression efficiency were examined. The results both in vivo and in vitro demonstrated that microbubble with ultrasound irradiation could significantly elevate the exogenous gene expression as compared with microbubble or ultrasound only. Overall, the present study showed that the ultrasound-target microbubble destruction method enhanced the exogenous gene expression in vivo and in vitro, and provided a gene therapy way not only efficient but also easy to be manipulated and carried out in clinical.

[Role of connective tissue growth factor in angiotensin II induced human proximal tubular cell hypertrophy].

OBJECTIVE: Renal cell hypertrophy is an important compensatory mechanism of chronic renal diseases, which has been shown closely correlated with the activation of intrarenal renin angiotensin system. However, the exact mechanism is still uncertain. The present study was to investigate the role of connective tissue growth factor (CTGF) in mediating the effect of angiotensin (AngII) induced human proximal tubular cell (HK-2) hypertrophy. METHODS: The cell line, HK-2, was grown in Dulbeccos's Modified Eagle's Medium (DMEM) containing 10% heat inactivated fetal calf serum (FCS). After rested in serum-free medium for 24 hours, The influence of anti-CTGF antibody on AngII induced cell protein de novo synthesis and total protein content were determined by [(3)H]-leucine incorporation and Coomassie brilliant blue G250 technique respectively. Fluorescence-activated cell sorter (FACS) flow cytometer was used to analyze the effect of anti-CTGF antibody on cell cycle distribution. The change of cellular size was determined by scanning electronic microscope (SEM). RESULTS: AngII significantly induced the increase of [(3)H] leucine incorporation in dose [AngII (mol/L): 0: 6926 +/- 1034; 10(-9): 8455 +/- 2137; 10(-7): 10 741 +/- 802; 10(-5): 12 945 +/- 1377] and time [AngII (10(-7)mol/L): 0 h: 5584 +/- 1016; 24 h: 7379 +/- 957; 48 h: 10 741 +/- 802; 72 h: 16 606 +/- 1177] dependent manner. Meanwhile, the influence of AngII on cell total protein content showed the similar manner. Anti-CTGF antibody significantly inhibited the AngII induced above effects dose and time dependently. 48 h after the stimulation by AngII (10(-7)mol/L), the percentage of cells in G0-G1 phase (76.09% +/- 1.82%) and the average cell diameter (20.6 microm +/- 3.8 microm) was significantly increased compared to the control (62.1% +/- 2.5%, 11.9 microm +/- 1.6 microm, P < 0.01 respectively), which could markedly reversed by treatment with anti-CTGF antibody (71.68% +/- 1.78%; 16.4 microm +/- 3.2 microm, P < 0.05, 0.01 respectively vs AngII group). CONCLUSION: AngII could induce the development of tubular cell hypertrophy, which might be mediated by CTGF.

[Expression of lecithin cholesterol acyltransferase and/or apoA-I mediated by recombinant adeno-associated virus in myogenic cells].

Lecithin cholesterol acyltransferase (LCAT) is the major enzyme producing most plasma cholesterol esters(CE) and a key participant in the process of reverse cholesterol transfer (RCT). The aim of this research is to co-express LCAT and it's natural activator apoA-I, with the recombinant adeno-associated virus vectors in the skeletal muscle cells, in order to pave a new way for gene therapy of the primary or secondary LCAT deficiency. 293T cells was cotransfected with pDG and rAAVAIL/rAAVL plasmids to produce infectious rAAV, and non-ionic iodixanol gradient centrifugation, followed by heparin affinity chromatography, were performed for separation, purification and concentration of rAAV. The particle numbers of rAAV, assayed by dot blot, were 7 x 10(14)/L (rAAVAIL) and 1 x 10(14)/L (rAAVL). These vectors were then transduced into C2C12 myoblasts. The results of ELISA and Western blot for human apoA-I, and [3H]-cholesterol-labeled radiochemical methods for LCAT activity, showed that the expression of human apoA-I cDNA and/or human LCAT cDNA in transduced C2C12 cells lasted for 30 days, even after myoblasts were differentiated into myotubes. PCR products for the transgene indicated the long-term persistence of transduced vector sequences. The results indicate that the methods used for production and purification of rAAV is efficient, and rAAV vector mediated the expression and secretion of LCAT and apoA-I gene in C2C12 myoblasts successfully. It suggests that the use of rAAV vectors mediating the high efficiency, long-term expression of human LCAT cDNA and/or apoA-I cDNA in skeletal muscle in vivo can be a safe and fesible strategy for the gene therapy of LCAT deficiency.

Inhibitory effects of praeruptorin C on cattle aortic smooth muscle cell proliferation.

AIM: To study the effects of praeruptorin C (pra-C) on proliferation of cattle aortic smooth muscle cells (SMC). METHODS: The DNA synthesis of SMC was measured using the incorporation of [3H]thymidine([3H]TdR). Cell cycle phase was evaluated by flow cytometry and cytotoxicity was evaluated by measuring lactic dehydrogenase (LDH) activity. RESULTS: Whether or not treated with angiotensin II (Ang-II), SMC proliferation was suppressed by pra-C in a concentration-dependent manner at range from 0.001 micromol/L to 10 micromol/L. The inhibitory effects appeared to be related to G1-S block in cell cycle traverse while the LDH activities did not change dramatically. CONCLUSION: Pra-C can completely inhibit SMC proliferation induced by Ang II and partly inhibit the growth of SMC- induced by bovine serum, which is important in prevention and treatment of vascular hyperplastic disease.

Inhibition of Scavenger Receptor A Expression Treated with PMA by the Inhibitor of Tyrosine Protein Kinase Genistein.

In order to investigate the relationship between scavenger receptor type A and cell signal transduction, human U937 macrophages were treated with tyrosine protein kinase inhibitor genistein, then the cells were incubated with [(125)I]ox-LDL or ox-LDL, and the cellular degradation of [(125)I]ox-LDL or binding were measured separately. Then the effect of the drug on cell surface expression of SR-A were measured by means of autoradiography, it was found that genistein could reduce cellular SR-A mRNA transcription by RT-PCR. The results indicated that genistein could reduce U937 macrophages to bind lipids and reduce SR-A expression by suppression of transcription, and could reduce degradation of lipids by U937 macrophage and accumulation of cholesterol within the cells. It suggests that the function of scavenger receptors may be correlated with cell tyrosine protein kinase, the mechanism is transcriptional and it also suggests that SR-A may participate in the signal transduction directly.

A Novel Oxidized Low Density Lipoprotein-binding Protein in Macrophage.

Scavenger receptor A(SR-A)on the mouse peritoneal macrophages(MPM) mediates the endocytic uptake of oxidized low density lipoproteins(ox-LDL). However, using ligand blotting and immunoblotting, a novel macrophage membrane protein binding to ox-LDL with estimated molecular mass of 92 kD was found. This membrane protein could not bind to ac-LDL. Its binding to ligands was not affected by reducing reagents either. Preincubation with medium containing neuraminidase dramatically decreased binding of the 92 kD membrane protein to ox-LDL. Excess amounts of ox-LDL could completely block the binding of (125)I ox-LDL to 92 kD membrane protein, but polyI, dextran sulfate and fucoidin showed partial competitive inhibition effects to the binding. These results suggest that the novel 92 kD membrane protein plays important role in the MPM uptake of ox-LDL.

Effect of Protein Kinase C Inhibitor on Scavenger Receptor in Human U937 Macrophage-like Cells.

In order to investigate effects of cell protein phosphorylation on scavenger receptor, human U937 macrophage-like cells were treated with protein kinase C inhibitor staurosporine, then the cells were incubated with (125)I ox-LDL or ox-LDL, and the cellular degradation of (125)I ox-LDL, its binding to receptor and the internalization of cell surface ox-LDL receptor complex as well as the accumulation of lipids within cells were measured separately. Moreover, the effects of the drug on expression of cell surface receptor were observed by means of autoradiography. The results indicated that staurosporine could enhance U937 cells to bind lipids and stimulate scavenger receptor expression, and could reduce degradation of lipids by U937 cells and the accumulation of cholesterol within the cells. It suggests that the function of scavenger receptors may be correlated with cell protein phorsphorylation.