ABSTRACT
Pigment epithelium-derived factor (PEDF) is a multifunctional protein that exhibits anti-angiogenic, antitumor, anti-inflammatory, antioxidative, anti-atherogenic, and cardioprotective properties. While it was recently shown that PEDF expression is inhibited under low oxygen conditions, the functional role of PEDF in response to hypoxia/reoxygenation (H/R) remains unclear. The goal of this study was to therefore investigate the influence of PEDF on myocardial H/R injury. For these analyses, PEDF-specific small interfering RNA-expressing and PEDF-expressing lentivirus (PEDF-LV) vectors were utilized to knockdown or stably overexpress PEDF, respectively, within human cardiomyocytes (HCM) in vitro. We noted that reactive oxygen species (ROS) play important roles in the induction of cell death pathways, including apoptosis and autophagy in ischemic hearts. Our findings demonstrate that overexpression of PEDF resulted in a significant reduction in ROS production and attenuation of mitochondrial membrane potential depletion under H/R conditions. Furthermore, PEDF inhibited the activation of a two-step apoptotic pathway in which caspase-dependent (caspase-9 and caspase-3) and caspase-independent (apoptosis inducing factor and endonuclease G), which in turn cleaves several crucial substrates including the DNA repair enzyme poly (ADP-ribose) polymerase. Meanwhile, overexpression of PEDF also promoted autophagy, a process that is typically activated in response to H/R. Therefore, these findings suggest that PEDF plays a critical role in preventing H/R injury by modulating anti-oxidant and anti-apoptotic factors and promoting autophagy.
Subject(s)
Eye Proteins/genetics , Eye Proteins/metabolism , Mitochondria/physiology , Myocytes, Cardiac/cytology , Nerve Growth Factors/genetics , Nerve Growth Factors/metabolism , Reactive Oxygen Species/metabolism , Serpins/genetics , Serpins/metabolism , Apoptosis , Autophagy , Caspases/metabolism , Cell Hypoxia , Cell Survival , Cells, Cultured , Gene Expression Regulation , Gene Knockdown Techniques , Humans , Membrane Potential, Mitochondrial , Models, Biological , Myocytes, Cardiac/metabolismABSTRACT
UNLABELLED: Swimming is a lifestyle intervention recommended by many clinicians in the prevention and treatment of hypertension. Yet, not all studies have agreed that swimming training can reduce blood pressure (BP). Inclusion of normotensive subjects could be a confounder for discrepancies among studies. In this one-year longitudinal study, long-term effects of swimming training on BP were investigated in 7 mild hypertensive patients (systolic BP (SBP) > 140 mmHg) and 16 normotensive controls. At baseline, these subjects (aged 21.5 +/- 0.1 years) did not participate in any form of sport training activity for the previous 3 months before enrollment into the training program. The training distance progressed from 0 (baseline) to 7 kilometers per week. BP and the homeostasis model assessment for insulin resistance (HOMA-IR) were determined under fasted condition at baseline and 48 h after the last swimming bout. The hypertensive patients displayed significantly greater HOMA-IR than age-matched normotensive controls. When data of all subjects were pooled, plasma glucose concentration was only slightly lowered after training, but weight, height, body mass index, SBP, diastolic BP (DBP) and HOMA-IR values were not significantly altered. However, when observation was restricted to the hypertensive patients, swimming training significantly lowered SBP by approximately 17 mmHg, concurrent with 41% reduction in HOMA-IR. Intriguingly, SBP in the normotensive subjects was elevated by approximately 6 mmHg after training. CONCLUSIONS: The present study found normalization rather than universal reduction effect of swimming training on BP. Furthermore, the BP-lowering effect of training in hypertensive patients appears to be associated with improvement in insulin sensitivity.
Subject(s)
Blood Pressure/physiology , Hypertension/physiopathology , Insulin Resistance/physiology , Swimming/physiology , Blood Glucose/metabolism , Exercise/physiology , Exercise Therapy , Humans , Hypertension/therapy , Longitudinal Studies , Treatment Outcome , Young AdultABSTRACT
Terminal transferase-dependent polymerase chain reaction (TDPCR) can be used after reverse transcription (RT) to analyze RNA. This method (RT-TDPCR) has been used for study of RNA structure and RNA-protein interactions at nucleotide-level resolution. A detailed protocol of RT-TDPCR is presented here with examples of its use with ribonuclease T1 in mammalian cells for detecting (1) RNA structure and protein footprints of the human ferritin heavy-chain messenger RNA and (2) in vivo structure of exon 4 of human XIST (X chromosome inactivation-specific transcript) RNA.
Subject(s)
Nucleotidyltransferases/metabolism , Proteins/metabolism , RNA/metabolism , Reverse Transcriptase Polymerase Chain Reaction/methods , Base Sequence , Cell Line, Tumor , Humans , Models, Genetic , Molecular Sequence Data , Nucleic Acid Conformation , Protein Binding , Proteins/chemistry , Proteins/genetics , RNA/chemistry , RNA/geneticsABSTRACT
The UV components of sunlight (UVA and UVB) are implicated in the etiology of human skin cancer. The underlying mechanism of action for UVB carcinogenicity is well defined; however, the mechanistic involvement of UVA in carcinogenesis is not fully delineated. We investigated the genotoxicity of UVA1 versus UVB in the overall genome and in the p53 tumor suppressor gene in normal human skin fibroblasts. Immuno-dot blot analysis identified the cis-syn cyclobutane pyrimidine-dimer (CPD) as a distinctive UVB-induced lesion and confirmed its formation in the genomic DNA of UVA1-irradiated cells dependent on radiation dose. HPLC/tandem MS analysis showed an induction of 8-oxo-7,8-dihydro-2'-deoxyguanosine in the genomic DNA of UVA1-irradiated cells only. Mapping of DNA damages by terminal transferase-dependent PCR revealed preferential, but not identical, formation of polymerase-blocking lesions and/or strand breaks along exons 5-8 of the p53 gene in UVB- and UVA1-irradiated cells. The UVB-induced lesions detected by terminal transferase-PCR were almost exclusively mapped to pyrimidine-rich sequences; however, the UVA1-induced lesions were mapped to purine- and pyrimidine-containing sequences along the p53 gene. Cleavage assays with lesion-specific DNA repair enzymes coupled to ligation-mediated PCR showed preferential, but not identical, formation of CPDs along the p53 gene in UVB- and UVA1-irradiated cells. Additionally, dose-dependent formation of oxidized and ring-opened purines and abasic sites was established in the p53 gene in only UVA1-irradiated cells. We conclude that UVA1 induces promutagenic CPDs and oxidative DNA damage at both the genomic and nucleotide resolution level in normal human skin fibroblasts.
Subject(s)
DNA Damage/genetics , Genes, p53/radiation effects , Genome, Human , Ultraviolet Rays , Cells, Cultured , Chromatography, High Pressure Liquid , DNA Repair Enzymes/metabolism , Dose-Response Relationship, Radiation , Genes, p53/genetics , Humans , Immunoblotting , Mass Spectrometry , Polymerase Chain Reaction/methods , Pyrimidine Dimers/radiation effectsABSTRACT
Terminal transferase-dependent PCR (TDPCR) can be used after reverse transcription to analyze RNA. This method (RT-TDPCR) is able to provide in vivo information at nucleotide-level resolution, and has been used for study of ribozymes, RNA size, RNA structure, and RNA-protein interactions. A detailed protocol of RT-TDPCR is presented here with examples of its use in detecting ribozyme cleavage intermediates in yeast and a RNA transcription start site in mammalian cells.
