Angiogenesis in fetal tendon development: spatial and temporal expression of the angiogenic peptide vascular endothelial cell growth factor

Abstract.

In traction tendons, whose line of action corresponds to that of the muscle, few blood vessels are uniformly distributed within the tendon tissue. In gliding tendons, which change their direction of pull, an avascular zone is normally found in the region where the tendon wraps around the pulley. This avascular fibrocartilaginous gliding zone is predisposed for degenerative changes and spontaneous rupture. Since factors regulating angiogenesis in tendons are largely unknown, we analyzed the expression of the vascular endothelial growth factor (VEGF) and its receptors VEGFR-1 (flt-1) and VEGFR-2 (KDR) in human fetal and adult tendon tissue by immunohistochemical, biochemical, and molecular biology methods. In order to elucidate whether mechanical stress might influence VEGF expression in tendon tissue we loaded primary cultures of rat tenocytes with intermittent hydrostatic pressure in a special cell culture chamber (amplitude: 0.2 Mpa, frequency: 0.1 Hz, time period: 5 h/day) and measured VEGF expression using ELISA. In fetal tendons high VEGF levels could be quantified by ELISA, whereas negligible ones were found in adult tissue. VEGF could be immunostained in tenocytes and endothelial cells. In the tibialis posterior tendon – as an example for a gliding tendon – VEGF immunostaining decreased in the gliding zone adjacent to the bony hypomochlion between week 20 and week 24 after gestation. This region remained largely avascular during the fetal period. In the peritendineum and in regions proximally and distally of the gliding zone immunostaining for VEGF was positive and factor VIII-positive microvessels could be detected. In these vessels, the VEGFR-1 (flt-1) and the VGEFR-2 could also be visualized. Reverse transcription-polymerase chain reaction (RT-PCR) confirmed the results regarding VEGF expression and showed further that the splice variants VEGF₁₂₁ and VEGF₁₆₅ are expressed exclusively during angiogenesis in fetal tendons. Monolayer cultures of tendon cells released measurable amounts of VEGF. Application of intermittent hydrostatic pressure decreased VEGF expression significantly. Thus, the angiogenic peptide VEGF is present in human fetal tendons, which are exposed to traction, but not in the avascular zone of gliding tendons, which are predominantly exposed to compressive and shearing forces. These findings support the view that the development of avascular zones in tendons might be caused by a mechanically induced downreglation of VEGF expression.