Inhibition of AXUD1 attenuates compression-dependent apoptosis of cementoblasts.

OBJECTIVES: Root resorptions are common undesirable side effects of orthodontic treatment. In most patients, these defects are repaired by cementoblasts. However, in 1-5 % of patients, the repair fails. The repair mechanism is not well understood. Apoptosis of cementoblasts might contribute to an impaired repair of root resorptions induced by orthodontic forces. MATERIALS AND METHODS: To gain insight into putative molecular pathways leading to compression-induced apoptosis of human primary cementoblasts (HPCBs), three independent cell populations were subjected to compressive loading at 5, 20, and 30 g/cm2 for 1, 6, and 10 h. The mRNA expression of AXUD1, a novel pro-apoptotic gene, was monitored by quantitative reverse transcription PCR (qRT-PCR). To identify a possible function in compression-dependent apoptosis, AXUD1 was silenced in cementoblasts using an siRNA approach. Apoptosis of cementoblasts was measured by annexin V staining and flow cytometry. The phosphorylation of c-Jun-N-terminal kinases (JNKs) was investigated by Western blotting. RESULTS: AXUD1 was significantly induced in a time- and force-dependent manner. The rate of apoptotic HPCBs increased by 20-40 % after 10 h of compression (30 g/cm2). Phosphorylation of JNKs was detected after 10 h at 30 g/cm2. SiRNA-mediated knockdown of AXUD1 led to decreased phosphorylation of JNKs and reduced apoptosis rates in compressed HPCBs. CONCLUSIONS: Compression-induced apoptosis of HPCBs is mediated by AXUD1 via a JNK-dependent pathway. CLINICAL RELEVANCE: AXUD1-dependent apoptosis of human cementoblasts might contribute to an impaired repair of root resorptions during orthodontic tooth movement. Further studies are needed to develop treatment strategies aiming to minimize root resorption during orthodontic tooth movement.

Development and validation of a novel cell-based assay for potency determination of human parathyroid hormone (PTH).

Parathyroid hormone (PTH) is the primary regulator of serum calcium homeostasis and plays a major role in bone metabolism. Its actions are mediated via the PTH1 receptor (PTH1R) resulting in adenylate cyclase activation and consequently production of cyclic adenosine mono-phosphate (cAMP). The latter stimulates cellular metabolic pathways. This study describes the development, validation and applications of a novel cell-based potency assay for PTH using HEK293 cells over-expressing PTH1R. PTH concentration-dependent cAMP formation in these cells was quantitatively analyzed employing time-resolved fluorescence technology (TR-FRET). The optimized assay was precise, reproducible and exhibited a high sensitivity to PTH with a limit of quantification in the low picogram range. The potencies of differently manufactured PTH1-34 peptides, as well as a full-length variant (PTH1-84), were all accurately measured. Since PTH activity is inhibited by neutralizing antibodies against PTH, the assay was adapted to detect and measure neutralizing antibodies in human serum. Thus, applications of this novel cell-based PTH potency assay were extended to immunogenicity testing of PTH preparations in non-clinical and clinical settings.

Vascular endothelial growth factor concentration in chronic subdural hematoma fluid is related to computed tomography appearance and exudation rate.

Chronic subdural hematoma (CSH) is characterized by a net increase of volume over time. Major underlying mechanisms appear to be hemorrhagic episodes and a continuous exudation, which may be studied using labeled proteins to yield an exudation rate in a given patient. We tested the hypothesis that the concentration of vascular endothelial growth factor (VEGF) in hematoma fluid correlates with the rate of exudation. Concentration of VEGF was determined in 51 consecutive patients with CSH by the sandwich immune enzyme-linked immunosorbent assay technique. Mean values were correlated with exudation rates taken from the literature according to the appearance of CSH on computed tomography (CT) images. The CT appearance of each CSH was classified as hypodense, isodense, hyperdense, or mixed density. Mean VEGF concentration was highest in mixed-density hematomas (22,403±4173 pg/mL; mean±standard error of the mean; n=27), followed by isodense (9715±1287 pg/mL; n=9) and hypodense (5955±610 pg/mL; n=18) hematomas. Only 1 patient with hyperdense hematoma fulfilled the inclusion criteria, and the concentration of VEGF found in this patient was 24,200 pg/mL. There was a statistically significant correlation between VEGF concentrations and exudation rates in the four classes of CT appearance (r=0.98). The current report is the first to suggest a pathophysiological link between the VEGF concentration and the exudation rate underlying the steady increase of hematoma volume and CT appearance.With this finding, the current report adds another piece of evidence in favor of the pathophysiological role of VEGF in the development of CSH, including mechanisms contributing to hematoma growth and CT appearance.

Short period of early reperfusion aggravates blood-brain barrier dysfunction during permanent focal ischemia in rats.

Unintentional reperfusion is considered a complication in various experimental models of focal brain ischemia. In the present study, we evaluated whether short intermittent reperfusion affects ischemic brain damage and blood-brain barrier (BBB) integrity in a model of permanent focal ischemia. Focal brain ischemia was induced in male Sprague-Dawley rats using the filament method. A 20-s reperfusion period was allowed 0.5, 2, or 10 min after thread occlusion of the middle cerebral artery. In control animals, the transient reperfusion episode was omitted. The infarct volume and extent of swelling was examined 24 h after permanent thread occlusion. Immunohistochemical staining for thrombin extravasation was performed. Transient reperfusion early after thread occlusion augmented brain swelling (control, 12.4 ± 8.5%; reperfusion after 0.5 min, 24.7 ± 7.0%*; after 2 min, 36.7 ± 4.8%*; after 10 min, 33.8 ± 4.9%*; *p < 0.01 vs. control) and significantly enhanced leakage of the plasma protein thrombin, whereas the ischemic volume was unaffected. Early intermittent reperfusion may be responsible for increased BBB disruption in permanent ischemia. Similar reperfusion episodes during early ischemia sequelae in patients-due to incomplete adherence or distal movements of a clot-may be causative for increased BBB damage, more severe edema, and potentially hemorrhagic transformation.

Angiotensin converting enzyme inhibition for arterial hypertension reduces the risk of recurrence in patients with chronic subdural hematoma possibly by an antiangiogenic mechanism.

OBJECTIVE: Chronic subdural hematoma (CSH) is characterized by pathological vascularization of the parietal membrane. Plasma leakage from immature vessels may be involved in hematoma enlargement and recurrence. We tested the hypothesis that the antiangiogenic side-effect of angiotensin converting enzyme (ACE)-inhibitor treatment for the control of arterial hypertension reduces the risk of recurrence in CSH. METHODS: We analyzed the data of 438 patients with CSH treated by a standard surgical procedure for hematoma evacuation in our department between 1995 and 2003. Patients with coagulopathies, malignancies, and independent neurological disorders were excluded from this study. Patient records were screened for age, sex, pre- and postoperative Markwalder score, arterial hypertension, medication with ACE-inhibitors, and recurrence of CSH. The rate of ACE-inhibitor treatment in our CSH patients was compared with an age-matched control group treated for herniated lumbar disc at the same time. The concentration of vascular endothelial growth factor was analyzed in hematoma samples and corresponding venous blood in 40 consecutive patients. RESULTS: A total of 310 patients were included in this study. The demographic data of Group A (with ACE-inhibition) and Group B (without ACE-inhibition) were comparable. In Group A, 5% (four out of 81) of the patients experienced recurrence as opposed to 18% (42 out of 229) in Group B (P = 0.00345). A negative correlation was found between the yearly rates of medication with ACE-inhibitors and recurrence (r = -0.8488; P = 0.0044). The rate of ACE-inhibitor treatment was lower in the CSH patients (25%) than in the control group (40%). The VEGF content was significantly lower in the hematoma in patients with ACE-inhibition (mean, 8891 pg/ml; range, 4300-18,300 pg/ml) than in patients without (mean, 22,565 pg/ml; range, 4200-89,650 pg/ml; P = 0.0116). CONCLUSION: Our data suggest that ACE-inhibitor treatment for the control of arterial hypertension lowers the risk of recurrence in patients undergoing operation for CSH and possibly even the development of CSH. This effect might be the result of an antiangiogenic mechanism of ACE-inhibitors.

Increased mRNA expression of VEGF within the hematoma and imbalance of angiopoietin-1 and -2 mRNA within the neomembranes of chronic subdural hematoma.

The aim of the study was to determine the source of vascular endothelial growth factor (VEGF) in hematoma fluid of patients suffering from chronic subdural hematoma (CSH) and to identify the level of gene expression of the pro-angiogenic factors angiopoietin 1 (ANG-1) and ANG-2 in hematoma membranes. Samples of venous blood, hematoma fluid, and outer membrane were obtained during surgery for CSH. The numbers of mononuclear cells were determined in hematoma fluid and in venous blood samples taken from 11 patients. The concentration of VEGF was measured by ELISA technique in hematoma fluid and in plasma. RT-PCR methodology was used to study the expression of different mRNA species in 11 patients. The mRNA species analyzed include VEGF and its receptors, VEGFR-1 and VEGFR-2, and ANG-1, ANG-2 and their receptor, Tie-2. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as housekeeping gene and was used for semiquantitative analysis. The VEGF concentration was several hundred fold higher in the hematoma fluid than in corresponding plasma samples. A significant correlation was found between the number of neutrophils and the VEGF content in the hematoma fluid. The expression levels of VEGF, mainly VEGF165 and VEGF121 mRNA were highest in cells obtained from the hematoma fluid. In membrane samples, mRNA encoding for VEGF and its receptors was only inconsistently detected while the mRNA species encoding for ANG-1, ANG-2, and Tie-2 were found throughout all samples. The mean ratio of ANG-1/ANG-2 mRNA expression was 0.48 as opposed to 1.9 in a normal human brain tissue sample. The results suggest that the hematoma cells are the primary source of VEGF. A marked increase in the expression of ANG-2 mRNA over ANG-1 mRNA demonstrates a pro-angiogenic pattern in the hematoma membranes. Persistent activation of the ANG/Tie-2 system in addition to high levels of VEGF may keep the vasculature in a destabilized condition and may account for the continuous formation of new and immature blood vessels resulting in massive plasma extravasation and repeated bleeding episodes. Thus, the present study provides new evidence in favor of pro-angiogenic mechanisms playing an important role in the pathophysiology of CSH.