2011年 · 第124卷第01期

BackgroundSusceptibility to coronary artery disease (CAD) and diabetes is encoded by distinct, tightly-linked single nucleotide polymorphisms on chromosome 9p21. This study aimed to examine the association of variant rs1333049 on chromosome 9p21.3 with early-onset and severity of CAD in Chinese patients with and without type 2 diabetes, and to determine the possible impact of rs1333049 on glucose metabolism and inflammation pathways.MethodsGenotyping of variant rs1333049 on chromosome 9p21.3 was performed in 2387 patients with and without diabetes who were undergoing coronary angiography to evaluate suspected or established CAD. Serum levels of glucose, glycosylated hemoglobin A1c (HbA1c), insulin, high-sensitivity C-reactive protein, tumor necrosis factor-α, and interleukin-6 were also measured, and compared with each patient’s genotype.ResultsThe homozygous CC genotype of rs1333049 was significantly associated with CAD in diabetic (OR: 1.270, P=0.044) and non-diabetic (OR: 1.369, P=0.011) patients after adjusting for traditional risk factors. There was an association between CC genotype and number of diseased vessels in diabetics (P=0.019), but not in non-diabetics (P=0.126). Among diabetic patients, CC genotype carriers had an increased risk of early-onset CAD (OR: 2.367, P=0.008) and greater cumulative atherosclerotic burden compared with non-CC genotype carriers (Gensini score: 31.80±17.20 vs. 23.09±21.63, P=0.039). No significant differences were observed between genotypes of rs1333049 in serum levels of glucose, insulin, HbA1c, or inflammatory cytokines for diabetic or non-diabetic patients with CAD.ConclusionsThis study demonstrated a significant association of rs1333049 polymorphism on chromosome 9p21.3 with CAD in Chinese diabetic and non-diabetic patients. The homozygous CC genotype of rs1333049 confers a magnified risk of early-onset and more severe CAD in diabetic patients through a novel biological pathway unrelated to glucose metabolism or inflammation.

BackgroundIt has been found that cardiac protection afforded by ischemic preconditioning (IPC) is significantly reduced in the senescent myocardium. ADAMTS-1 (a disintesrin and metalloprotease with thrombospondin type 1 motifs) has been shown to inhibit angiogenesis in a variety of in vitro and in vivo assays. The aim of this study was to investigate the age-associated differences in ADAMTS-1 protein expression in rat myocardium after ischemic preconditioning.MethodsSixty-four young (4 months) and old (24 months) male Sprague-Dawley rats were randomly assigned to an IPC group (40 rats) or a sham group (rats). A model of delayed IPC was induced and rats were sacrificed and myocardial samples were harvested from the ischemic-reperfused region for immunohistochemical detection of ADAMTS-1 at serial time points after IPC. A model of myocardial infarction was produced by ligation of the left anterior descending coronary artery in additional sets of young and old rats after sham or IPC procedures, then age-associated myocardial infarction survival after IPC was calculated.ResultsADAMTS-1 expression increased significantly in old rats compared to young rats (P<0.05). The mean densities of ADAMTS-1 protein at 0, 6, 12, and 24 hours in young-IPC group after IPC were 0.05±0.01, 0.13±0.03, 0.16±0.04, and 0.12±0.03 vs. 0.07±0.03, 0.20±0.03, 0.24±0.05, and 0.21±0.04 in old-IPC group. IPC resulted in diminished survival rates (5/35 vs. 6/14, old-IPC group vs. old-sham group, P<0.05), reduced left ventricular fractional shortening ((13.9±2.8)% vs. (18.3±2.3)%, P<0.05) and increased the myocardial infarction size ((37.9±3.2)% vs. (32.8±5.1)%, P<0.05) in the older rats.ConclusionsCardioprotection with IPC is attenuated in the older heart. ADAMTS-1 expression induced by IPC is greater in old rats. Over-expression of anti-angiogenic factors might be a potential mechanism behind reduced protection after IPC associated with aging.

BackgroundThe microemboli produced during spontaneous plaque rupture and ulceration and during coronary intervention will reduce coronary reserve and cause cardiac dysfunction. It is though that inflammation caused by the microinfarction induced by the microembolization may play an essential role. It is known that the activation of p38 mitogen-activated protein kinases (MAPK) in both infected and non-infected inflammation in myocardium may cause a contractile dysfunction. But the relation between the activation of p38 MAPK and microembolization is still unknown.MethodsSprague-Dawley rats were randomly divided into three groups: Sham group, coronary microembolization (CME) group and SB203580 group (n=10 per group). CME rats were produced by injection of 42 μm microspheres into the left ventricle with occlusion of the ascending aorta. SB203580, a p38 MAPK inhibitor, was injected into the femoral vein after the injection of microspheres to make the SB203580 group. Left ventricular ejection fraction (LVEF) was determined by echocardiography. The protein concentration of P38 MAPK in the myocardium was assessed by Western blotting. The relative expression of mRNA for tumor necrosis factor (TNF)-α was assessed by the technique of semi-quantitative polymerase chain reaction amplification.ResultsLVEF was depressed at three hours up to 12 hours in the CME group. Increased p38 MAPK activity and TNF-α mRNA expression were observed in the CME group. The administration of SB203580 partly inhibited p38 MAPK activity, but did not fully depress the TNF-α expression, and partly preserved cardiac contractile function.Conclusionsp38 MAPK is significantly activated by CME and the inhibition of p38 MAPK can partly depress the TNF-α expression and preserve cardiac contractile function.

BackgroundRNA interference (RNAi) is a potential cure for amyotrophic lateral sclerosis (ALS) caused by dominant, gain-of-function superoxide dismutase 1 (SOD1) mutations. The success of such therapy relies on the functional small interfering RNAs (siRNAs) that can effectively deliver RNAi. This study aimed to design the functional siRNAs targeting ALS-associated mutant alleles.MethodsA modified dual luciferase system containing human SOD1 mRNA target was established to quantify siRNA efficacy. Coupled with validated siRNAs identified in the literature, we analyzed the rationale of siRNA design and subsequently developed an asymmetry rule-based strategy for designing siRNA. We then further tested the effectiveness of this design strategy in converting a naturally symmetric siRNA into functional siRNAs with favorable asymmetry for gene silencing of SOD1 alleles.ResultsThe efficacies of siRNAs could vary tremendously by one base-pair position change. Functional siRNAs could target the whole span of SOD1 mRNA coding sequence as well as non-coding region. While there is no distinguishable pattern of the distribution of nucleobases in these validated siRNAs, the high percent of GC count at the last two positions of siRNAs (P18 and P19) indicated a strong effect of asymmetry rule. Introducing a mismatch at position 1 of the 5′ of antisense strand of siRNA successfully converted the inactive siRNA into functional siRNAs that silence SOD1 with desired efficacy.ConclusionsAsymmetry rule-based strategy that incorporates a mismatch into siRNA most consistently enhances RNAi efficacy and guarantees producing functional siRNAs that successfully silence ALS-associated SOD1 mutant alleles regardless target positions. This strategy could also be useful to design siRNAs for silencing other disease-associated dominant, gain-of-function mutant genes.

BackgroundHypoxia-inducible factor (HIF) may play an important role in the process of tumorigenesis as well as tumor progression. The aim of this study was to compare the expression between HIF-1α and HIF-2α in tumor angiogenesis and the overall impact on patient prognosis in human non-small cell lung cancer (NSCLC).MethodsIn the current work we compared the immunohistochemical expression of HIF-1α and HIF-2α in surgical specimens of 140 patients with NSCLC in a tissue microarray study. Relationships between HIF-α expression and clinicopathological or angiogenic factors, including prognosis, were analyzed.ResultsHigh HIF-1α and HIF-2α expression was noted in 49/140 (35.0%) and in 64/140 (45.7%) of the cases, respectively. There was no direct correlation between HIF-1α and HIF-2α expression. Patients with advanced stage tumors had frequent high expression of HIF-2α (P=0.007), and we also found a significant correlation between HIF-2α and T or N stage (P=0.030 and 0.043, respectively). HIF-1α showed a marginal association with T stage (P=0.084), which showed a higher expression in early stage tumors. A significant correlation (P=0.045) was noticed between HIF-1α and vascular endothelial growth factor (VEGF) expression while the expression levels of thymidine phosphorylase (TP), cyclooxygenase (COX)-2 and microvessel density (MVD) were significantly higher in high HIF-2α tumors (P=0.020, 0.004, and 0.046, respectively). In addition, univariate analysis of overall survival demonstrated that HIF-2α expression, but not HIF-1α, was related to poor outcome (P=0.001) and it retained significant in multivariate analysis (P=0.036).ConclusionsTaken together, we conclude that HIF-1α and HIF-2α may differentially regulate the major angiogenic factors in different stages of the tumor process in NSCLC. HIF-2α may play a dominant role in tumor angiogenesis and appears to be of obvious value as a significant prognostic factor in NSCLC.

BackgroundThe hairpin cell-penetrating peptides (hCPPs) demonstrate an interesting characteristic of conditioned activation by molecules. We hypothesized that hCPPs have the potential to selectively deliver a paramagnetic gadolinium probe into the matrix metalloproteinase 2 (MMP-2) positive human ovary adenocarcinoma cell lines, SKOV-3.MethodshCPPs were synthesized and labeled with 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid gadolinium (III) (Gd-DOTA) and fluorescein isothiocyanate (FITC) by f-moc strategy using a standard solid phase peptide synthesis protocol. MMP-2 expression and activity were demonstrated by reverse transcriptase polymerase chain reaction (RT-PCR) and zymography. Internalization and location of hCPPs in SKOV-3 cells were observed by fluorescein imaging and flow cytometery. Selective delivery of Gd-DOTA in SKOV-3 cells was observed by magnetic resonance imaging (MRI) and transmission electron microscopy (TEM).ResultsThe uptake of hCPPs by SKOV-3 cells depended on the activity of MMP-2. T1WI signals of SKOV-3 cells treated with Gd-DOTA-hCPPs suggested the uptake of Gd-DOTA-hCPPs increased in a time- (r=0.990, P<0.01) and concentration-dependent manner (r=0.964, P<0.001), but was inhibited by a MMP-2 inhibitor. Electron-dense particles observed in the cytoplasm and nucleus by transmission electron microscopy proved the intracellular penetration of gadolinium.ConclusionshCPPs can be used as an effective vector for an MRI molecular probe to assess the activity of MMP-2.

BackgroundDelta-like 4 (DLL4) is an endothelium specific Notch ligand and has been shown to function as a regulating factor during physiological and pathological angiogenesis. It has been reported that the DLL4-Notch signaling pathway is regulated by hypoxia and may prevent excessive angiogenesis through the inhibition of angiogenic branching and by triggering vessel maturation. Choroidal neovascularization (CNV) is a pathological form of angiogenesis in which hypoxia is thought to play an important role. This study was aimed to evaluate the role of DLL4 in the development of CNV.MethodsWe utilized chemical hypoxia induced by 200 μmol/L CoCl2 to observe the expression of DLL4 in choroid-retinal endothelial cells (RF/6A cells), which are the primary cells involved in CNV. After transfection of a DLL4 small interfering RNA (siRNA), mRNA and protein expression of DLL4 and key downstream genes, including HES1 and HEY1, in hypoxic RF/6A cells were investigated by RT-PCR, real-time PCR, and Western blotting analysis. Three controls were used: one without transfection, one with transfection reagent, and one with scrambled negative control siRNA. The effects of the DLL4 siRNA on the biological function of hypoxic RF/6A cells during angiogenesis, including cell proliferation, migration and tube formation, were investigated.ResultsThe results showed that hypoxic conditions led to upregulation of DLL4 expression in RF/6A cells in vitro. After transfection, siRNA-duplex1 targeting DLL4 depleted the DLL4 mRNA levels by as much as 91.4% compared with the scrambled siRNA control, and DLL4 protein expression was similarly effected. There was no significant difference in DLL4 expression among the blank control, transfection reagent control, and scrambled siRNA groups. In addition, after transfection of hypoxic RF/6A cells with the DLL4 siRNA-duplex1, the mRNA levels ofHES1andHEY1, which function downstream of DLL4-Notch signaling, were lowered by 75.1% and 86.3%, respectively, compared with the scrambled siRNA control. Furthermore, knockdown of DLL4 expression significantly promoted the proliferation of hypoxic RF/6A cells and led to their arrest in the S phase of the cell cycle. Migration and tube formation of hypoxic RF/6A cells were significantly induced by the DLL4 siRNA, with the number of migrated cells increased by 1.6-fold and total tube length increased by 82.3%, compared with the scrambled siRNA (P<0.05).ConclusionsDLL4 functions as a negative regulator of angiogenic branching and sprouting. Based on our results, DLL4 signaling appears to play an essential role in the biological behavior of choroid vascular endothelial cells under hypoxia. Therefore, DLL4 may represent a novel target for CNV therapy in the future.

BackgroundHepatocyte growth factor (HGF) treats ischemic disease by promoting arteriogenesis, however, its mechanism of action is not known. The notch signaling pathway plays an important role in neovascularization. The relationship between the proliferation and migration ability of artery endothelial cells and the Dll4-Notch-Hey2 signaling pathway in the process of arteriogenesis was investigated as a mechanism of action of HGF.MethodsBased on the prophase study cells and supernatant were harvested at the indicated time after human femoral artery endothelial cells (HFAECs) were infected with adenovirus-HGF (Ad-HGF) at 200 pfu/cell. Cells were analyzed for HGF expression and Notch1, Dll4 and Hey2 expression by ELISA and reverse transcription-PCR (RT-PCR). The changes in the proliferation and migration ability of HFAECs were observed by MTT and Transwell migration experiments. Ad-GFP-infected HFAECs were used as control.ResultsCompared with the control group the Ad-HGF group’s HGF expression was not increased with time, and the induction by HGF of Notch1, Dll4 and Hey2 gene transcription was not enhanced with an increase of HGF. The proliferation ability of Ad-HGF-transduced HFAECs was enhanced and their migration ability was also enhanced in the presence of HGF.ConclusionsThrough activating the Dll4-Notch-Hey2 signaling pathway, HGF indirectly promotes the proliferation and migration ability of cells, so that offspring artery branches are formed.

BackgroundMolecular targeted drugs is now widely used in non-small cell lung cancer (NSCLC) clinical treatment. Icotinib hydrochloride is a new type of oral epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKIs). In this study, we examined the role of EGFR, K-RAS, B-RAF somatic mutations and EGFR mRNA expression in tumor specimens from advanced NSCLC patients as predicators of the efficacy of icotinib hydrochloride.MethodsWe analyzed tumor paraffin-embedded specimens, which were obtained from 14 of 40 patients with advanced NSCLC who enrolled in the stage I clinical trial of icotinib hydrochloride. Somatic mutations were evaluated by mutant-enriched liquidchip (MEL) technology, and EGFR mRNA expression was measured by branched DNA liquidchip (MBL) technology.ResultsIn the 14 specimens, seven patients showed EGFR mutations, exon 19 deletion (3/7) and exon 21 point mutation (4/7); and two patients showed K-RAS mutation. No mutations in EGFR exon 20 or B-RAF were detected. In patients with EGFR mutation, one patient developed progress disease (PD), three patients had stable disease (SD), two patients had partial responses (PR) and one patient had a complete response (CR). In patients with wild-type EGFR, four patients had PD, three patients acquired SD, and none had PR/CR (P=0.0407). EGFR mutations were associated with better progress-free survival (PFS) (141 days vs. 61 days) but without a statistically significant difference (P=0.8597), and median overall survival (OS) (≥449 days vs. 140 days). EGFR mRNA expression levels were evaluated (three high, eight moderate, one low, and two that can not be measured due to insufficient tumor tissue) and no statistically significant relationships was observed with response, PFS or OS.ConclusionsThe EGFR mutation rate was consistent with that reported in the Asian population, so the MEL technology is reliable for measuring EGFR mutation with high throughput and rapidity. EGFR exon 19 deletions and exon 21 point mutation are predictive biomarkers for response to icotinib hydrochloride as second line treatment or above.

BackgroundThe signal transducer and activator of transcription 6 (STAT6) expression in lung epithelial cells plays a pivotal role in asthma pathogenesis. Activation of STAT6 expression results in T helper cell type 2 (Th2) cell differentiation leading to Th2-mediated IgE production, development of allergic airway inflammation and hyperreactivity. Therefore, antagonizing the expression and/or the function of STAT6 could be used as a mode of therapy for allergic airway inflammation.MethodsIn this study, we synthesized a 20-mer phosphorothioate antisense oligonucleotide (ASODN) overlapping the translation starting site of STAT6 and constructed STAT6 antisense RNA (pANTI-STAT6), then transfected them into murine spleen lymphocytes and analyzed the effects of antagonizing STAT6 functionin vitroand in a murine model of asthma.ResultsIn vitro, we showed suppression of STAT6 expression and interleukin (IL)-4 production of lymphocytes by STAT6 ASODN. This effect was more prominent when cells were cultured with pANTI-STAT6. In a murine model of asthma associated with allergic pulmonary inflammation in ovalbumin (OVA)-sensitized mice, local intranasal administration of fluorescein isothiocyanate (FITC)-labeled STAT6 ASODN to DNA uptake in lung cells was accompanied by a reduction of intracellular STAT6 expression. Such intrapulmonary blockade of STAT6 expression abrogated signs of lung inflammation, infiltration of eosinophils and Th2 cytokine production.ConclusionThese data suggest a critical role of STAT6 in the pathogenesis of asthma and the use of local delivery of STAT6 ASODN as a novel approach for the treatment of allergic airway inflammation such as in asthma.

Ceftriaxone-induced immune haemolytic anemia is rare but severe complication of this type of antibiotics. In this article, we present a 43-year old patient who suffered from ceftriaxone-induced haemolysis complicated with acute portal vein thrombosis. After successful salvage and transfusion, we underwent thrombolysis via superior mesenteric artery route. Totally recanaliztion achieved. Repeated CT venography showed portal vein still patent with 6 months oral anti coagulation treatment.

BackgroundGlycemic variability, an HbA1c-independent risk factor, has more deleterious effects than sustained hyperglycemia in the development of diabetic complications. This study analyzed the characteristics of glycemic variability in type 2 diabetes mellitus (T2DM) with HbA1c<6.5% in duration of twice daily premixed insulin treatment and the effect of further treatment with acarbose.MethodsEighty-six T2DM patients who used premixed insulin analogue (insulin aspart 30) twice daily and had HbA1c<6.5% and 20 controlled subjects with normal glucose regulation (NGR) were monitored using the continuous glucose monitoring (CGM) system. The mean amplitude of glycemic excursions (MAGE), mean of daily differences (MODD) were used for assessing intra-day, inter-day glycemic variability. Hypoglycemia was defined as glucose level<3.9 mmol/L for at least 15 minutes in CGM. According to reference values of MAGE, T2DM patients were classified into two groups: low-MAGE group with MAGE<3.4 mmol/L (L-MAGE) and high-MAGE group with MAGE ≥3.4 mmol/L (H-MAGE). H-MAGE group received further treatment with acarbose for 2 weeks and was monitored a second time with CGM system.ResultsAfter first CGM, L-MAGE group had 41 cases, and H-MAGE group had 45 cases. The MAGE and MODD of T2DM group were all higher than those of subjects with NGR (P<0.01). Twenty-four percent (n=11) in H-MAGE group had a total of 13 hypoglycemic events, 10 of the 13 events occurred at night, meanwhile 5% (n=2) in L-MAGE group had a total of 2 hypoglycemic events, which also occurred at night (hypoglycemic events: 24% vs. 5%, χ2=6.40, P<0.01). MAGE value was correlated with hypoglycemia value and 2-hour postprandial plasma glucose value (r=–0.32 and 0.26, respectively, P<0.05). After further acarbose therapy and secondly CGM, MAGE and MODD values in H-MAGE group were all significantly decreased (40%,P<0.01, and 15%, P<0.05, respectively), but remained higher than in the subjects with NGR (P<0.05); 2% (n=1) had a total of 1 hypoglycemic event, incidence significantly decreased (2% vs. 24%, χ2=9.61, P<0.01).ConclusionsCGM system can detect the glycemic variability and asymptomatic hypoglycemic events of T2DM with well-controlled HbA1c in duration of insulin treatment. Combination therapy of premixed insulin twice daily with acarbose can flat glycemic variability and decrease hypoglycemic events.

BackgroundEndometriosis (EM) is a benign gynecologic disease predominantly found in women of reproductive age. However, its pathogenesis is still poorly understood. Our experiment was designed to establish a stable and reliable cultural environment for coculture of endometrium and peritoneum, so as to observe the adhesion/invasion ability of endometrium from patients with or without EM.MethodsEndometria of secretory phase and peritoneum were sampled from 6 women with endometriois during laparoscopy. Six with ovarian teratoma or simple ovarian cyst were taken as control. We cocultured endometrium and peritoneum into four groups (endometrium from EM cultured with peritoneum from EM, endometrium from control cultured with peritoneum from control, endometrium from EM cultured with peritoneum from non-EM and the endometrium from control cultured with peritoneum from EM) to observe the adhesion/invasion process in gas-liquid surface culture and in-medium culture. Specimens were collected at 1 hour, 6 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days and 7 days for histology, immunofluorescence and immunohistochemical analysis on cytokeratin 8 (CK8) and CD10.ResultsThe gas-liquid surface culture was superior to in-medium culture for the maintenance of tissue morphology and survival of endometrium. CK8 immunoflurescence demonstrated no remarkable difference in adhesion process between patients with and without EM. CD10 immunochemistry manifested frequent invasion of endometrial stromal cells from EM patients into peritoneum of up to 3 days culture, while the endometriotic cells from non-EM patients did not invade into peritoneum.ConclusionsGas-liquid surface culture is a suitable model for observing the early events in EM lesion formation. Endometrium from patients with EM showed increased invasion capacity during coculture, which might help to explain the etiology of endometriosis.

Retroperitoneal hemangiopericytoma is a kind of uncommon tumor. We report a case of 41-year-old man who was diagnosed retroperitoneal tumor without significant symptoms by abdominal ultrasonography. Abdominal CT and MRI scans show a 6 cm × 5 cm solid tumor delineated clearly from adjacent organs. Excision of the tumor was performed and the histopathological examination confirmed the diagnosis of hemangiopericytoma.

ObjectiveTo elucidate the development of mapping and localization of susceptible genes on chromosomes to asthma related phenotypes.Data sourcesPublished articles about susceptibility genes for asthma related phenotypes were selected using PubMed.Study selectionUsing methodsof candidate gene positional clone and genome-wide scan with linkage and association analysis to determine the location in the genome of susceptibility genes to asthma and asthma related phenotypes.ResultsThere are multiple regions in the genome harboring susceptibility genes to asthma and asthma related phenotypes, including chromosomes 5, 11, 12, 6, 2, 3, 13, 7, 14, 9, 19 and 17. Many of these regions contain candidate genes involved in asthma development and progression. Some susceptible genes may affect the phenotype expression or response to therapy. In addition, the interaction of multiple genes with the environment may contribute to the susceptibility to asthma.ConclusionsAs an essential step toward cloning the susceptible genes to asthma, fine mapping and localization on chromosomes are definitely needed. Novel powerful tools for gene discovery and the integration of genetics, biology and bioinformatics should be pursued.