Effect of post-exercise muscle cooling on PGC-1α and VEGF mRNA expression in Thoroughbreds

Besides preventing exertional heat illness, muscle cooling can be a potential strategy to enhance exercise-training induced adaptations. This study aimed to examine the effects of post-exercise cooling on the mRNA expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and vascular endothelial growth factor (VEGF) in Thoroughbred skeletal muscle. Five Thoroughbred horses performed treadmill running until their pulmonary artery temperature reached 42 °C, followed by walking on the treadmill with no additional cooling (CONT) or muscle cooling with a shower using the tap water (26 °C, 0.4 l/s; COOL), for 30 min. Muscle biopsies were obtained before (PRE) and 3 h after exercise (3 Hr-REC) from the gluteus medius muscle. PGC-1α mRNA expression was elevated 3 h after exercise in both the CONT (PRE vs 3 Hr-REC: 1.0±0.1 vs 5.0±0.8, P<0.01) and COOL (PRE vs 3 Hr-REC: 1.1±0.3 vs 6.6±0.9, P<0.01) conditions; however, there was no difference between the two conditions at 3 h after exercise (P=0.17). VEGF mRNA expression was elevated 3 h after exercise in COOL (PRE vs 3 Hr-REC: 1.0±0.2 vs 2.2±0.2, P<0.05) but not in CONT (PRE vs 3 Hr-REC: 1.0±0.1 vs 1.8±0.3, P=0.08). VEGF mRNA expression at 3 h after exercise was significantly negatively correlated with rectal temperature at the end of the 30-min cooling period (r = -0.65, P<0.05). Our results suggest that the decline in body temperature after exercise may lead to greater expression of the key angiogenic gene in Thoroughbred horses.

Endothelial cAMP deactivates ischemia-reperfusion-induced microvascular hyperpermeability via Rap1-mediated mechanisms

Approaches to reduce excessive edema due to the microvascular hyperpermeability that occurs during ischemia-reperfusion (I/R) are needed to prevent muscle compartment syndrome. We tested the hypothesis that cAMP-activated mechanisms actively restore barrier integrity in postischemic striated muscle. We found, using I/R in intact muscles and hypoxia-reoxygenation (H/R, an I/R mimic) in human microvascular endothelial cells (HMVECs), that hyperpermeability can be deactivated by increasing cAMP levels through application of forskolin. This effect was seen whether or not the hyperpermeability was accompanied by increased mRNA expression of VEGF, which occurred only after 4 h of ischemia. We found that cAMP increases in HMVECs after H/R, suggesting that cAMP-mediated restoration of barrier function is a physiological mechanism. We explored the mechanisms underlying this effect of cAMP. We found that exchange protein activated by cAMP 1 (Epac1), a downstream effector of cAMP that stimulates Rap1 to enhance cell adhesion, was activated only at or after reoxygenation. Thus, when Rap1 was depleted by small interfering RNA, H/R-induced hyperpermeability persisted even when forskolin was applied. We demonstrate that 1) VEGF mRNA expression is not involved in hyperpermeability after brief ischemia, 2) elevation of cAMP concentration at reperfusion deactivates hyperpermeability, and 3) cAMP activates the Epac1-Rap1 pathway to restore normal microvascular permeability. Our data support the novel concepts that 1) different hyperpermeability mechanisms operate after brief and prolonged ischemia and 2) cAMP concentration elevation during reperfusion contributes to deactivation of I/R-induced hyperpermeability through the Epac-Rap1 pathway. Endothelial cAMP management at reperfusion may be therapeutic in I/R injury. NEW & NOTEWORTHY Here, we demonstrate that 1) stimulation of cAMP production deactivates ischemia-reperfusion-induced hyperpermeability in muscle and endothelial cells; 2) VEGF mRNA expression is not enhanced by brief ischemia, suggesting that VEGF mechanisms do not activate immediate postischemic hyperpermeability; and 3) deactivation mechanisms operate via cAMP-exchange protein activated by cAMP 1-Rap1 to restore integrity of the endothelial barrier.

Expression of HIF-2αand VEGF in Cervical Squamous Cell Carcinoma and Its Clinical Significance

CSCC is a systemic disease involving polygenic alteration and multiple steps, and HIF and VEGF are closely associated with tumorigenesis. Specimens surgically resected from 64 cases of CSCC and 22 cases of normal cervical tissue were selected randomly to detect the expression of HIF-2αand VEGF in CSCC for exploring their clinical significance; information regarding the age, lymph node metastasis, and FIGO staging were collected as well; expression of HIF-2αand VEGF was detected by qPCR and immunohistochemistry. We found that the expression of HIF-2αand VEGF mRNA in CSCC was significantly higher than that of normal cervical tissues and showed a positive correlation between them. The positive rates of HIF-2αand VEGF protein expression in CSCC and normal cervical tissues were 93.8% and 18.2%, respectively, with correlation between them. The expression of both HIF-2αand VEGF mRNA did not relate closely to age but the FIGO staging and lymph node metastasis. Compared with the counterpart control group, CSCC tissues with high FIGO staging and lymph node metastasis had a higher level of HIF-2αand VEGF mRNA expression. So, HIF-2αand VEGF were overexpressed in CSCC, which has a great clinical significance for its diagnosis.

An oxidative DNA “damage” and repair mechanism localized in the VEGF promoter is important for hypoxia-induced VEGF mRNA expression

In hypoxia, mitochondria-generated reactive oxygen species not only stimulate accumulation of the transcriptional regulator of hypoxic gene expression, hypoxia inducible factor-1 (Hif-1), but also cause oxidative base modifications in hypoxic response elements (HREs) of hypoxia-inducible genes. When the hypoxia-induced base modifications are suppressed, Hif-1 fails to associate with the HRE of the VEGF promoter, and VEGF mRNA accumulation is blunted. The mechanism linking base modifications to transcription is unknown. Here we determined whether recruitment of base excision DNA repair (BER) enzymes in response to hypoxia-induced promoter modifications was required for transcription complex assembly and VEGF mRNA expression. Using chromatin immunoprecipitation analyses in pulmonary artery endothelial cells, we found that hypoxia-mediated formation of the base oxidation product 8-oxoguanine (8-oxoG) in VEGF HREs was temporally associated with binding of Hif-1α and the BER enzymes 8-oxoguanine glycosylase 1 (Ogg1) and redox effector factor-1 (Ref-1)/apurinic/apyrimidinic endonuclease 1 (Ape1) and introduction of DNA strand breaks. Hif-1α colocalized with HRE sequences harboring Ref-1/Ape1, but not Ogg1. Inhibition of BER by small interfering RNA-mediated reduction in Ogg1 augmented hypoxia-induced 8-oxoG accumulation and attenuated Hif-1α and Ref-1/Ape1 binding to VEGF HRE sequences and blunted VEGF mRNA expression. Chromatin immunoprecipitation-sequence analysis of 8-oxoG distribution in hypoxic pulmonary artery endothelial cells showed that most of the oxidized base was localized to promoters with virtually no overlap between normoxic and hypoxic data sets. Transcription of genes whose promoters lost 8-oxoG during hypoxia was reduced, while those gaining 8-oxoG was elevated. Collectively, these findings suggest that the BER pathway links hypoxia-induced introduction of oxidative DNA modifications in promoters of hypoxia-inducible genes to transcriptional activation.