Initial apoptosis is followed by increased proliferation of apoptosis-resistant endothelial cells

We have demonstrated that VEGF receptor blockade in combination with chronic hypoxia causes in rats severe angioproliferative pulmonary hypertension (SAPH) associated with arterial occlusion by proliferating endothelial cells, and we postulate that the established, lumen-occluding lesions are the result of the emergence of apoptosis-resistant proliferating cells. To study the dependence of exuberant endothelial cell proliferation on initial apoptosis, we adapted the CELLMAX artificial capillary system to analyze the effects of a VEGF receptor antagonist (SU5416) on human pulmonary microvascular endothelial cells under pulsatile shear stress. Immunohistochemical staining for caspase-3 and PCNA and flow cytometry for Annexin-V and BrdU supported our concept, since SU5416 caused initial apoptosis (35.8% at 24 h after the SU5416 addition and 4.8% in control cells) whereas the surviving cells became hyperproliferative (PCNA positive). Flow cytometry showed that apoptosis inhibition prevented the proliferation following the initial apoptosis. These lumen-filling endothelial cells were apoptosis resistant, grew without serum, and were phenotypically altered in that they express the tumor marker survivin. Hyperproliferative apoptosis-resistant cells were also generated by adding apoptosed cells instead of the VEGF receptor blocker to the CELLMAX system. In conclusion, endothelial cell death resulted in the selection of an apoptosis-resistant, proliferating phenotypically altered endothelial cell phenotype.