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.

Vimentin and laminin are altered on cheek pouch microvessels of streptozotocin-induced diabetic hamsters

OBJECTIVE: Normal endothelial cells respond to shear stress by elongating and aligning in the direction of fluid flow. Hyperglycemia impairs this response and contributes to microvascular complications, which result in deleterious effects to the endothelium. This work aimed to evaluate cheek pouch microvessel morphological characteristics, reactivity, permeability, and expression of cytoskeleton and extracellular matrix components in hamsters after the induction of diabetes with streptozotocin. METHODS: Syrian golden hamsters (90-130 g) were injected with streptozotocin (50 mg/kg, i.p.) or vehicle either 6 (the diabetes mellitus 6 group) or 15 (the diabetes mellitus 15 group) days before the experiment. Vascular dimensions and density per area of vessels were determined by morphometric and stereological measurements. Changes in blood flow were measured in response to acetylcholine, and plasma extravasation was measured by the number of leakage sites. Actin, talin, α-smooth muscle actin, vimentin, type IV collagen, and laminin were detected by immunohistochemistry and assessed through a semiquantitative scoring system. RESULTS: There were no major alterations in the lumen, wall diameters, or densities of the examined vessels. Likewise, vascular reactivity and permeability were not altered by diabetes. The arterioles demonstrated increased immunoreactivity to vimentin and laminin in the diabetes mellitus 6 and diabetes mellitus 15 groups. DISCUSSION: Antibodies against laminin and vimentin inhibit branching morphogenesis in vitro. Therefore, laminin and vimentin participating in the structure of the focal adhesion may play a role in angiogenesis. CONCLUSIONS: Our results indicated the existence of changes related to cell-matrix interactions, which may contribute to the pathological remodeling that was already underway one week after induction of experimental diabetes.

Betamethasone inhibits tumor development, microvessel density and prolongs survival in mice with a multiresistant adenocarcinoma TA3

Tumor resistance to traditional cancer treatments poses an important challenge to modern science. Thus, angiogenesis inhibition is an important emerging cancer treatment. Many drugs are tested and corticosteroids have shown interesting results. Herein we investigate the effect on microvessel density, survival time and tumoral volume of mice with TA3-MTX-R tumors. Twenty six mice were inoculated with lxlO6 tumor cells, 4-5 days after injection, six mice were injected with PBS (group A) and twenty mice were treated with p-met (group B). All animals from Group A died on day 22. Group B was divided into Bl (treated discontinued) and B2 (treated daily) and observed until day 88. All mice were processed for histo-immunohistochemical analysis and the blood vessels were counted. A decrease in microvessel density and tumoral volume and longer survival times were observed in the treated group. We propose that the antiangiogenic p-met effect explains, at least partially, its tumor inhibitory properties. As an important perspective, we will experimentally combine these strategies with those recently described by us with regard to the important antiangiogenic-antitumor effects of Trypanosoma cruzi calreticulin. Since the molecular targets of these strategies are most likely different, additive or synergic effects are envisaged.