Endothelial Membrane Remodeling Is Obligate for Anti-Angiogenic Radiosensitization during Tumor Radiosurgery

Abstract

BackgroundWhile there is significant interest in combining anti-angiogenesis therapy with conventional anti-cancer treatment, clinical trials have as of yet yielded limited therapeutic gain, mainly because mechanisms of anti-angiogenic therapy remain to a large extent unknown. Currently, anti-angiogenic tumor therapy is conceptualized to either “normalize” dysfunctional tumor vasculature, or to prevent recruitment of circulating endothelial precursors into the tumor. An alternative biology, restricted to delivery of anti-angiogenics immediately prior to single dose radiotherapy (radiosurgery), is provided in the present study.Methodology/Principal FindingsGenetic data indicate an acute wave of ceramide-mediated endothelial apoptosis, initiated by acid sphingomyelinase (ASMase), regulates tumor stem cell response to single dose radiotherapy, obligatory for tumor cure. Here we show VEGF prevented radiation-induced ASMase activation in cultured endothelium, occurring within minutes after radiation exposure, consequently repressing apoptosis, an event reversible with exogenous C16-ceramide. Anti-VEGFR2 acts conversely, enhancing ceramide generation and apoptosis. In vivo, MCA/129 fibrosarcoma tumors were implanted in asmase+/+ mice or asmase−/− littermates and irradiated in the presence or absence of anti-VEGFR2 DC101 or anti-VEGF G6-31 antibodies. These anti-angiogenic agents, only if delivered immediately prior to single dose radiotherapy, de-repressed radiation-induced ASMase activation, synergistically increasing the endothelial apoptotic component of tumor response and tumor cure. Anti-angiogenic radiosensitization was abrogated in tumors implanted in asmase−/− mice that provide apoptosis-resistant vasculature, or in wild-type littermates pre-treated with anti-ceramide antibody, indicating that ceramide is necessary for this effect.Conclusions/SignificanceThese studies show that angiogenic factors fail to suppress apoptosis if ceramide remains elevated while anti-angiogenic therapies fail without ceramide elevation, defining a ceramide rheostat that determines outcome of single dose radiotherapy. Understanding the temporal sequencing of anti-angiogenic drugs and radiation enables optimized radiosensitization and design of innovative radiosurgery clinical trials.

Regulation of Neuronal Stem Cell Proliferation in the Hippocampus by Endothelial Ceramide

“…Finally, several studies demonstrated that Vascular Endothelial Growth Factor (VEGF), which promotes endothelial cell proliferation and stabilizes endothelial cells [45,46], acts anti-depressive. Since VEGF inhibits the acid sphingomyelinase [47], it is possible that this anti-depressive effect of VEGF is related to inhibition of the acid sphingomyelinase/ ceramide system in endothelial cells. However, neuronal stem cells express VEGF-receptor 2 [48] and it is therefore also possible that VEGF does not only act on endothelial cells but also directly on neuronal stem cells after crossing the blood-brain barrier.…”

Anti-ceramide antibody prevents the radiation gastrointestinal syndrome in mice

“…BAECs were cultured in DMEM, switched at confluency to DMEM plus 2% normal calf serum for 1-week minimum (8), and preincubated for 18 hours before irradiation in DMEM plus 0.2% human albumin.…”

“…While conventional radiobiology considers unrepaired or misrepaired DNA doublestrand breaks in stem cell clonogens (SCCs) as autonomous lesions leading to irreversible tissue injury, our recent studies have challenged this paradigm, presenting genetic evidence that acute endothelial damage also plays a major role in GI tract injury (4)(5)(6). Within minutes of radiation exposure, endothelial acid sphingomyelinase (ASMase) is activated, catalyzing ceramide generation on the external plasma membrane of mouse and human endothelium to initiate apoptotic signaling (7,8). Endothelium displays 20-fold more ASMase than other mammalian cells, almost exclusively in a secretory form, which makes them particularly vulnerable to ceramide-induced apoptosis (9,10).…”

Combining angiogenesis inhibition and radiotherapy: A double-edged sword

“…The angiostatic agents were only radiosensitizing when delivered immediately prior to single dose radiotherapy (<1 h). Other schedules, applying the angiostatic drugs 24 h or 48 h prior to radiotherapy, or directly after the radiotherapy had no additional anti-tumor effect (Truman et al, 2010). The anti-tumor effect of blocking the deltalike 4 (Dll4)/Notch pathway after radiotherapy was studied in Dll4 expression or Dll4 non-expressing cancer cell lines as xenografts in mice.…”