Circulating endothelial progenitor cells from patients with renal cell carcinoma display aberrant VEGF regulation, reduced apoptosis and altered ultrastructure

Abstract

Endothelial colony forming cells (ECFCs) are the only endothelial progenitor cells (EPCs) subtype belonging to the endothelial phenotype and capable of forming neovessels in vivo. We have recently shown that the intracellular Ca2+ machinery plays a key role in ECFC activation and is remodeled in ECFCs isolated from patients suffering from renal cellular carcinoma (RCC-ECFCs). More specifically, ECFCs upregulate the store-operated Ca2+ entry (SOCE) machinery, while they seemingly show a reduction in the Ca2+ concentration within the endoplasmic reticulum ([Ca2+]ER). Metastatic RCC patients are commonly treated with an anti-vascular endothelial growth factor (VEGF) therapy, but they show either intrinsic or adaptive refractoriness, which ultimately leads to their death. Herein, we assessed whether and how the rearrangement of the Ca2+ machinery impacts on the pro-angiogenic Ca2+ response to VEGF, which stimulates normal ECFCs (N-ECFCs) through an oscillatory Ca2+ response. We found that VEGF stimulates the nuclear translocation of p65/RelA, a major component of the Ca2+-dependent transcription fac- tor NF-kB, in N-ECFCs. This process is blocked by the pharmacological abrogation of VEGF-induced Ca2+ oscillations. We further showed that NF-kB controls VEGF-induced protein expression of E-selectin, VCAM-1 and MMP9. Likewise, VEGF-induced expression was also inhibited by the pharmacological suppression of the accompanying Ca2+ spikes. Thus, VEGF induces a Ca2+-dependent, NF-kB-mediated protein expression in N-ECFCs. VEGF did not trigger protein expression in RCC-ECFCs despite the fact that VEGFR-2 was normally expressed and auto-phosphorylated. Our subsequent studies employed the tar- geted recombinant Ca2+-sensitive photoprotein aequorin to confirm that [Ca2+]ER is lower in RCC-ECFCs; surprisingly, electron microscopy analysis revealed that the endoplasmic reticulum cisternae are enlarged rather than shrinked in these cells. These results show for the first time that VEGF fails to stimulate tumor-derived ECFCs: these findings could therefore help to understand the relative failure of anti-VEGF treatment in RCC patients. References