Immunhistochemische Untersuchung von Integrinen auf Endothelzellen in Abhängigkeit von der extrazellulären Matrix und von verschiedenen stimulierenden Substanzen
In this study we examined the expression of integrins on human venous endothelial cells (HUVEC) after adhesion to different matrix proteins by immunofluorescence microscopy. Additionally, we compared the influence of several stimulating factors (Phorbol-12-Myristate-13-Acetate/ PMA, Forskolin, Basic Fibroblast Growth Factor/ BFGF, Vascular Endothelial Growth Factor/ VEGF) on the expression of integrins. In the second part of the study we investigated the changes of cell morphology and integrin distribution after incubation with fibrin and after co-incubation with fibrin and the stimulating factors. Endothelial cells (EC) represent an important functional part of the vascular system. They actively participate in inflammatory reactions, hemostasis and in the formation of new blood vessels (angiogenesis). EC express integrin receptors on their cell membrane that promote their attachment to subendothelial matrix proteins. After contact with adhesive proteins these integrin molecules are clustered in focal adhesions (FA). The focal contacts depend on the composition of the extracellular matrix (ECM). Our data show that EC adhere to fibronectin via the alpha 5 beta 1 integrin and to vitronectin and fibrin via the alpha v beta 3 integrin. EC on vitronectin produce endogenous fibronectin which they secrete as an additional matrix protein and to which they adhere via alpha 5 beta 1 integrins. After activation of proteinkinase C (PKC) by PMA we see an increased alpha v beta 3-integrin-expression on fibronectin as well as on vitronectin. After stimulation with forskolin we find a temporary loss of the typical FA pattern on both matrix proteins. Stimulation with angiogenic growth factors (BFGF, VEGF) for 12 hours does not influence the expression of alpha 5 beta 1- and alpha v beta 3 integrins. After contact with a fibrin clot EC retract and the normal cobblestone monolayer architecture of cultured vascular endothelium is disrupted. This process is mediated by integrins. They integrate the activities of the ECM and the cytoskeleton. According to the substratum specifity fibrin leads to an increased expression of alpha v beta 3 integrins. Stimulation with PMA detains the fibrin-induced cell retraction. The alpha 5 beta 1 - and alpha v beta 3 integrins are not transferred into the cytoplasma as we can see after a sole incubation with fibrin. After co-stimulation of EC with fibrin and angiogenic growth factors (BFGF, VEGF) we do not see an alteration of integrin expression compared to the respective single incubation. In conclusion, a modified ECM can change the integrin expression within a short period of time. In our test series with a maximum period of stimulation of 12 hours the angiogenic growth factors VEGF and BFGF do not alter the integrin pattern of HUVEC. Integrin-blocking antibodies are supposed to influence neoangiogenic processes.
info:eu-repo/classification/ddc/610, Medizin
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