The effect of hirudin on antagonisting thrombin induced apoptosis of human microvascular endothelial cells

Abstract Purpose: To investigate whether hirudin exerts its antithrombin action to decrease the ratio of Human Microvascular Endothelial Cells (HMVECs) apoptosis. Methods: Human microvascular endothelial cells (HMVECs) cultured in the third and fifth generations were used. HMVECs were divided into normal group, thrombin group (T group), natrual hirudin group (H group), thrombin + natrual hirudin group (T + H group), AG490 group, thrombin + AG490 group (T + AG490 group), natrual hirudin + AG490 group (H + AG490 group), thrombin + natural hirudin + AG490 (T + H + AG490 group).Apart from the normal group, the other groups were exposed to the relevant drugs for 24 hours.HMVEC apoptosis was assessed by flow cytometric and double Immunofluorescence of phosphorylation of JAK (P-JAK2) and TUNEL assay. Results: Compared with the normal group, in thrombin group the HMVECs apoptosis rate were significantly increased (P<0.05).The results indicated that the index of apoptosis and the apoptosis rate were improved in cultures treated by natural hirudin (T + H group), relative to cultures with thrombin only (T group). We found that the index of apoptosis and the apoptosis rate in the AG490 + thrombin group were higher than that in the hirudin + thrombin group (P<0.05). Double Immunofluorescence of p-JAK2 and TUNEL assays showed that cells were double positive for P-JAK2 uptake and TUNEL detection liquid binding. Conclusion: The natural hirudin and JAK2/STATs signal inhibitor AG490 could block the effects of thrombin. Natural hirudin could attenuate HMVECs apoptosis via antagonizing thrombin and it is suggested that this effect may occur by blocking the JAK2/STATs signaling pathway and this signaling pathways appears to be not the only pathway.

Assessment of Mean Vessel Density and Angiogenesis in Endometrial Carcinomas.

Objective: To evaluate the role of angiogenesis in tumor growth by the assessment of mean vessel density and to quantify angiogenesis as an important variable in endometrial cancers. Material and Methods: 53 cases of endometrial malignancies (epithelial tumors-36 cases and metastatic tumors-17 cases), were analysed for histological types, grades and features like depth of invasion and vascular invasion. Microvessel counts were performed by examining the microvessels thoroughly in terms of count, morphology and density after staining the tissues by hematoxylin & eosin stain, reticulin and immunostain (Antifactor VIII Ag). Results: On H&E stain - Microvessel density (MVD) in endometrial malignancy ranged from 3.0 - 13.5 and mean MVD was 8.78. On Reticulin stain - MVD ranged from 3.5 - 15.2 and mean MVD was 9.76. Antifactor VIII sections showed very small microvessels or even single endothelial cells with the highest total counts and the MVD ranged from 6.5- 16.8 with Mean MVD of 11.7. The counts increased with the grade of the tumor in the absence of necrosis or haemorrhage. MVD counts also increased with the stage, being 8.12 in Stage I disease, 8.65 in Stage II and 10.8 in stage III disease. Atypical hyperplasia was found to be associated with epithelial tumors in 8 cases, making it a significant finding. Conclusion: Role of angiogenesis assumes greater significance with increasing severity of lesions, higher grade and stage of the tumor and seems to have an important diagnostic and prognostic significance.

Relation of CD117 immunoreactivity and microvascular density in invasive breast carcinoma.

Background and Objective: In breast cancer, the expression of CD117 represents a highly controversial subject but the majority of studies have found decreased c-kit expression in malignant breast epithelium. A number of studies have reported that increased intratumoral microvessel density (MVD) is associated with poor prognosis in breast cancer. The aim of the study was to assess the relation of CD117 and MVD with other clinicopathological parameters in invasive breast carcinomas using the tissue microarray technique. Materials and Methods: A total of 126 cases of invasive breast carcinoma of different histological types and grades were collected from files of a pathology department during 2010. Clinicopathological and histological parameters were evaluated. Sections from formalin-fixed, paraffin-embedded tumor tissues microarray blocks were immunostained with CD117 and CD34. Statistical analysis of data was done using SPSS, version 16.0. Results: About 29% of invasive breast carcinomas were CD117 positive. There were significant differences between expression of CD117 in the tumor epithelial cells and age of the patient; tumor grade; tumor size, and LN metastasis. Also, there was significant relation between expression of CD117 in the tumor epithelial cells and MVD, expression of estrogen, and progesterone receptors. On multivariate analysis, the most important predictors of negativity of CD117 were tumor size and positive lymph node involvement. Conclusion: Lack of CD117 immunoreactivity in invasive breast carcinoma was associated with features of more aggressive tumor behavior as higher microvessel density, larger size, higher tumor grade, more lymph node metastasis, and negative estrogen and progesterone receptors.

The Effect of Treadmill Exercise on Ischemic Neuronal Injury in the Stroke Animal Model: Potentiation of Cerebral Vascular Integrity

PURPOSE: This study was done to identify whether pre-conditioning exercise has neuroprotective effects against cerebral ischemia, through enhance brain microvascular integrity. METHODS: Adult male Sprague-Dawley rats were randomly divided into four groups: 1) Normal (n=10); 2) Exercise (n=10); 3) Middle cerebral artery occlusion (MCAo), n=10); 4) Exercise+MCAo (n=10). Both exercise groups ran on a treadmill at a speed of 15 m/min, 30 min/day for 4 weeks, then, MCAo was performed for 90 min. Brain infarction was measured by Nissl staining. Examination of the remaining neuronal cell after MCAo, and microvascular protein expression on the motor cortex, showed the expression of Neuronal Nuclei (NeuN), Vascular endothelial growth factor (VEGF) & laminin. RESULTS: After 48 hr of MCAo, the infarct volume was significantly reduced in the Ex+MCAo group (15.6+/-2.7%) compared to the MCAo group (44.9+/-3.8%) (p<.05), and many neuronal cells were detected in the Ex+MCAo group (70.8+/-3.9%) compared to the MCAo group (43.4+/-5.1%) (p<.05). The immunoreactivity of laminin, as a marker of microvessels and Vascular endothelial growth factor (VEGF) were intensively increased in the Ex+MCAo group compared to the MCAo group. CONCLUSION: These findings suggest that the neuroprotective effects of exercise pre-conditioning reduce ischemic brain injury through strengthening the microvascular integrity after cerebral ischemia.