Mechanical factors influence the expression of endostatin--an inhibitor of angiogenesis--in tendons

Abstract

Avascular zones of tendons are predisposed for degenerative changes and spontaneous rupture. Therefore, we analyzed the expression of the endogenous angiogenesis inhibiting factor endostatin in human fetal and adult tendons by immunohistochemical and biochemical methods. Moreover, to elucidate factors involved in the regulation of vascularity, we exposed primary cultures of rat tendon cells to intermittent hydrostatic pressure (0.2 MPa, 0.1 Hz for 24 h), and measured the endostatin content by ELISA and the effect of the conditioned medium to the proliferation of human umbilical vein endothelial cells (HUVEC). In fetal tendons high endostatin levels could be quantified by ELISA whereas low levels were found in adult tissue. In fetal tendons endostatin could also be immunostained in endothelial cells but mainly in fibroblasts. In adult Achilles tendons endostatin immunostaining was restricted to endothelial cells. In the tibialis posterior tendon--as an example for "wrap around"--endostatin immunostaining remained positive in the fibrocartilage adjacent to the medial malleolus. Fibrochondrocytes of the type II collagen positive fibrocartilage were intensively stained with the endostatin antibody. Factor VIII immunostaining showed that this region was largely avascular. Monolayer cultures of tendon cells released measurable amounts of endostatin into their culture supernatants. Application of intermittent hydrostatic pressure increased endostatin expression significantly. The conditioned media of tendon fibroblasts cultivated under intermittent hydrostatic pressure inhibited the proliferation of HUVEC in a dose dependent way. The spatial expression of endostatin in adult gliding tendons suggests that mechanical factors are involved in the regulation of this anti-angiogenic factor. In accordance, tendon cells exposed to intermittent hydrostatic pressure inhibit endothelial cell proliferation via humoral factors and produce endostatin. These findings support the view that the development and maintenance of avascular zones in tendons might be caused by a mechanically induced upregulation of anti-angiogenic factors.