Static, but not cyclic tensional forces on the heart increase anti-angiogenic sFlt-1 expression in endothelial cells and inhibit VEGFR-2 activation

Purpose: The left ventricle (LV) is subject to increased mechanical forces in a variety of diseases that cause chronic increased systolic or diastolic pressures. These are often associated with endothelial dysfunction and/or reduced numbers of capillaries. Further mechanical forces are known to modulate endothelial gene expression. Therefore, we hypothesized, that increase in static tensional forces on capillary endothelial cells of the LV result in up-regulation of soluble VEGF receptor 1 (sFlt-1) and thus leading to an imbalance of proand anti-angiogenic stimuli in the heart. Methods: A rabbit Langendorff heart with retrograde constant pressure perfusion was used as model. As perfusate oxygenated Dulbecco’s modified Eagle’s medium was used to keep the heart viable as long as possible without deterioration of its function or sings of ischemia. For the tensional force 5g per g of heart weight was applied to the apex of the beating LV either as static load or every 30 seconds (cyclic). For a set of experiments cardiac contractions were completely arrested using butanedione monoxime (BDM). Quantitative RT-PCR, immunoblots and co-immunoprecipitation was used to quantify mRNA and proteins of sFlt-1, vascular endothelial growth factor (VEGF) and VEGF receptor-2 (VEGFR-2). Immunohistochemistry was performed for colocalized of proteins. Results: Increased static tensional force on the LV leads to significant increase in mRNA (3,8 fold, p < 0.05) and protein expression of sFlt-1 (1,4 fold, p < 0.05), which can be detected as early as after 30min and remains increased for the duration of the experiments (up to 6h). Cyclic tensional forces, however, do not affect sFlt-1 mRNA or protein levels. VEGF levels are unaffected by both static and cycle forces on the beating heart. Complete cardiac arrest without affecting the endothelium by BDM, an ATPase inhibitor of skeletal myosin 2, does not affect sFlt-1 or VEGF expression. Increased sFlt-1 due to increased static force on the LV binds more VEGF, shown by co-immunoprecipitation of sFlt-1 and VEGF, and reduced VEGFR-2 phosphorylation. Endothelial cells of the heart were colocalized by immunohistochemistry as source of sFlt-1 release. Conclusions: Static, but not cyclic tensional forces on the LV result in increased release of anti-angiogenic sFlt-1 from capillary endothelial cells. While expression levels of VEGF remain unchanged, sFlt-1 binds VEGF and reduces activation of the signalling Receptor VEGFR-2. Thus, chronic increased forces in the LV contribute to the anti-angiogenic setting and reduced capillary density as seen in many heart diseases.