Objective: To construct a recombinant adeno-associated virus (AAV) vector harboring human hypoxia inducible factor-1α (HIF-1α) gene and to express it in primarily cultured neurons in the hippocampus. Methods: Human HIF-1α gene was inserted into AAV vector plasmid pSNAV 2.0 to generate a recombinant plasmid pSNAV-HIF-1α, then pSNAV-HIF-1α was transfected into BHK-21 cells and selected by G418. The construction of rAAV-HIF-1α was achieved by infecting G418-resistant BHK-21 cells with recombinant herpes simplex virus. The recombinant rAAV-HIF-1α was confirmed by polymerase chain reaction(PCR). The titer of rAAV-HIF-1α was detected by dot-blot with digoxin labelled cytomegalovirus probe. The purity of rAAV-HIF-1α was detected by SDS-PAGE. The expression of HIF-1α protein was determined by Western blot. Results: PCR demonstrated that human HIF-1α gene was successfully inserted into rAAV-HIF-1α. The Virus titer was 1 × 1012/ml and the virus purity exceeded 98%. Western nblot analysis demonstrated that rAAV-HIF1α infected hippocampal neurons expressed HIF-1α protein. Conclusion: rAAV-HIF-1α has been successfully constructed and it is capable of expressing HIF-1α in cultured hippocampal neurons.

OBJECTIVETo observe the effect of intracerebroventricular injection of rAAV-HIF-1α on hippocampal neuronal apoptosis in a rat model of Alzheimer disease (AD).

METHODSThirty-two male SD rats (250-300 g) were randomized into 4 groups (n=8), including the normal control group without any treatment, AD model group with right intracerebroventricular injection of 2 µl Aβ25-35 (10 mg/m1), sham-operated group with right intracerebroventricular injection of 2 µl normal saline, and AD+ rAAV-HIF-1α group with right intracerebroventricular injection of 10 µl rAAV-HIF-1a (1×10¹² v.g./m1) one week after Aβ25-35 injection. The rats were sacrificed to detect the expression of HIF-1α and apoptosis of hippocampal neurons 5 weeks after Aβ25-35 or saline injection.

RESULTSWestern blotting showed that the expression of HIF-1α was significantly higher in AD+rAAV-HIF-1α group (451.59±34.39) than in normal control group (229.05±41.28) and sham-operated group (216.29±37.08) (P<0.05) without significant difference between the latter two groups. The apoptotic ratio of the hippocampal neurons was significantly higher in AD model group ([19.49±2.59]%) than in normal control group ([5.41±0.75]%) and sham-operated group ([5.28±0.66]%) in (P<0.05), and intracerebroventricular injection of rAAV-HIF-1α resulted in a significant reduction of the apoptotic ratio in the AD rats ([12.07±2.06]%) (P<0.05).

CONCLUSIONIntracerebroventricular injection of rAAV-HIF-1α can inhibit hippocampal neuronal apoptosis in the rat model of AD.

Alzheimer Disease ; metabolism ; therapy ; Animals ; Apoptosis ; Disease Models, Animal ; Hippocampus ; cytology ; Hypoxia-Inducible Factor 1, alpha Subunit ; administration & dosage ; genetics ; metabolism ; Lateral Ventricles ; Male ; Neurons ; drug effects ; pathology ; Rats ; Rats, Sprague-Dawley

The study is to investigate the effect of angiotensin II (Ang II) and its receptor blockers on migration and endothelin-1 (ET-1) expression of rat vascular adventitial fibroblast subpopulations. Vascular adventitial fibroblasts were individually expanded by using cloning rings, and the effects of Ang II on the migration of adventitial fibroblast subpopulations were evaluated by Transwell. Fluorescence quantitative-PCR detected the expression of preproET-1 mRNA induced by Ang II, and its receptor antagonists losartan and PD-123319. The concentration of ET-1 was determined by ELISA. It showed that spindle shaped and epithelioid shaped cells were isolated by using cloning rings, named as spindle cells and round cells. RT-PCR showed that fibroblast subpopulations did not have leukocytes, endothelial cells and smooth muscle cells, namely pure cell lines. Compared with respective control cells, two subpopulations had transferring ability. Ang II significantly improved round cells migration in a concentration-dependent manner, and had no obvious influence on spindle cells migration. Ang II (1 x 10(-8) - 1 x 10(-6) mol x L(-1)) significantly increased the expression of preproET-1 mRNA in round cells (P < 0.01), and had no significant effect on the expression of preproET-1 mRNA in spindle cells. Losartan blocked the expression of preproET-1 mRNA induced by Ang II in round cells, and had no significant effect on the expression of preproET-1 mRNA in spindle cells. The effects of Ang II and ET-1 receptor inhibitors on the release of ET-1 were similar to the expression of preproET-1 mRNA. The results indicate that there are two cell subpopulations: round cells and spindle cells in rat vascular adventitial fibroblasts. Ang II significantly improved cells migration, and increased the expression of ET-1 in round cell subpopulation. It suggested that there may be different migratory mechanisms in two cell subpopulations, and the two subpopulations may play a different role in vascular remodeling and reparative process.
Angiotensin II ; pharmacology ; Angiotensin Receptor Antagonists ; pharmacology ; Animals ; Cell Movement ; drug effects ; Cells, Cultured ; Endothelin-1 ; genetics ; metabolism ; Fibroblasts ; cytology ; metabolism ; Imidazoles ; pharmacology ; Losartan ; pharmacology ; Male ; Pyridines ; pharmacology ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley ; Vasoconstrictor Agents ; pharmacology

BACKGROUNDThere is a paucity of studies investigating the clinical and biochemical characteristics of pain in chronic heart failure (CHF) patients. This study aimed to determine the clinical and biochemical characteristics and outcomes in Chinese patients with CHF and symptoms of pain.

METHODSSociodemographics, serum levels of creatinine, NT-proBNP, high-sensitivity C-reactive protein (hs-CRP), tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-10, and two-dimensional echocardiographic left ventricular ejection fraction (LVEF) were determined in 305 patients with CHF. A questionnaire packet including the Brief Pain Inventory (BPI) and the Minnesota Living with Heart Failure Questionnaire (MLHFQ) was used to assess the degree of pain rated on a 0 - 10 scale and the quality of life (QOL). A six-minute walking test was performed during routine clinic visits. Major adverse cardiac events (MACE) were recorded; including all-cause or cardiac mortality and rehospitalization because of myocardial infarction, worsening heart failure or stroke at follow-up.

RESULTSPain occurred in 25.6% of CHF patients, and was more common when the New York Heart Association (NYHA) functional class was worse. More patients with pain were female in gender, and had more co-morbidities, lower LVEF, and shorter distance during the 6-minute walking test. Despite similar serum levels of creatinine, N-terminal prohormone of brain natriuretic peptide (NT-proBNP), IL-6 and IL-10, the TNF-α levels were higher and MLHFQ scores were greater in CHF patients with pain. At follow-up, CHF patients with moderate to severe pain (≥ 4 scale) had higher rates of all-cause and cardiac mortality and rehospitalization because of myocardial infarction, worsening heart failure or stroke. Multivariate regression analysis revealed that the presence of pain was an independent risk factor for MACE and reduced QOL in CHF patients.

CONCLUSIONSPain occurs in all stages of the CHF trajectory, and its incidence increases as clinical functional status is worsened. The presence of pain exerts a negative impact on clinical outcome and QOL in patients with CHF.

C-Reactive Protein ; metabolism ; Echocardiography ; Female ; Heart Failure ; metabolism ; physiopathology ; Humans ; Interleukin-10 ; blood ; Interleukin-6 ; blood ; Male ; Pain ; metabolism ; physiopathology ; Tumor Necrosis Factor-alpha ; blood

BACKGROUNDPotentially lethal ventricular arrhythmias (PLVAs) occur frequently in survivors after acute myocardial infarction and are increasingly recognized in other forms of structural heart diseases. This study investigated the prevalence and prognostic significance of PLVAs in patients with chronic heart failure (CHF).

METHODSData concerning demographics, etiology of heart failure, NYHA functional class, biochemical variables, electrocardiographic and echocardiographic findings, and medical treatments were collected by reviewing hospital medical records from 1080 patients with NYHA II-IV and a left ventricular (LV) ejection fraction ≤ 45%. PLVAs were defined as multi-focal ventricular ectopy (> 30 beats/h on Holter monitoring), bursts of ventricular premature beats, and nonsustained ventricular tachycardia. All-cause mortality, sudden death, and rehospitalization due to worsening heart failure, or cardiac transplantation during 5-year follow-up after discharge were recorded.

RESULTSThe occurrence rate of PLVAs in CHF was 30.2%, and increased with age; 23.4% in patients < 45 years old, 27.8% in those between 45 - 65 years old, and 33.5% in patients > 65 years old (P = 0.033). Patients with PLVAs had larger LV size and lower ejection fraction (both P < 0.01) and higher all-cause mortality (P = 0.014) during 5-year follow-up than those without PLVAs. Age (OR 1.041, 95%CI 1.004 - 1.079, P = 0.03) and LV end-diastolic dimension (OR 1.068, 95%CI 1.013 - 1.126, P = 0.015) independently predicted the occurrence of PLVAs. And PLVA was an independent factor for all-cause mortality (RR 1.702, 95%CI 1.017 - 2.848, P = 0.031) and sudden death (RR 1.937, 95%CI 1.068 - 3.516, P = 0.030) in patients with CHF.

CONCLUSIONPLVAs are common and exert a negative impact on long-term clinical outcome in patients with CHF.

Adult ; Aged ; Arrhythmias, Cardiac ; mortality ; physiopathology ; Electrocardiography ; Female ; Heart Failure ; physiopathology ; Humans ; Male ; Middle Aged ; Regression Analysis