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Updated: Feb 26, 2026
A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors
Published on: March 15, 2016
Early Actions of Anti-Vascular Endothelial Growth Factor/Vascular Endothelial Growth Factor Receptor Drugs on
Basel Sitohy1, Sunghee Chang1, Tracey E Sciuto1
1The Center for Vascular Biology Research and the Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts.
Abstract:
Tumors induce their heterogeneous vasculature by secreting vascular endothelial growth factor (VEGF)-A. Anti-VEGF/VEGF receptor (VEGFR) drugs treat cancer, but the underlying mechanisms remain unclear. An adenovirus expressing VEGF-A (Ad-VEGF-A164) replicates the tumor vasculature in mice without tumor cells. Mother vessels (MV) are the first angiogenic vessel type to form in tumors and after Ad-VEGF-A164. Multiday treatments with a VEGF trap reverted MV back to normal microvessels. We now show that, within hours, a single dose of several anti-VEGF drugs collapsed MV to form glomeruloid microvascular proliferations (GMP), accompanied by only modest endothelial cell death. GMP, common in many human cancers but of uncertain origin, served as an intermediary step in MV reversion to normal microvessels. The vasodisruptive drug combretastatin CA4 also targeted MV selectively but acted differently, extensively killing MV endothelium. Antivascular changes were quantified with a novel Evans blue dye assay that measured vascular volumes. As in tumors, Ad-VEGF-A164 strikingly increased endothelial nitric oxide synthase (eNOS) expression. The eNOS inhibitor N(G)-Nitro-l-arginine methyl ester mimicked anti-VEGF/VEGFR drugs, rapidly collapsing MV to GMP. Inhibition of eNOS reduces synthesis of its vasodilatory product, nitric oxide, leading to arterial contraction. Patients and mice receiving anti-VEGF/VEGFR drugs develop hypertension, reflecting systemic arterial contraction. Together, anti-VEGF/VEGFR drugs act in part by inhibiting eNOS, causing vasocontraction, MV collapse to GMP, and subsequent reversion of GMP to normal microvessels, all without extensive vascular killing.
Insights
Anti-vascular endothelial growth factor (VEGF) drugs rapidly collapse tumor mother vessels into glomeruloid microvascular proliferations by inhibiting endothelial nitric oxide synthase (eNOS), leading to vessel normalization without extensive cell death.
Area of Science:
- Oncology
- Vascular Biology
- Pharmacology
Background:
- Tumors develop abnormal vasculature driven by vascular endothelial growth factor (VEGF)-A.
- Anti-VEGF/VEGF receptor (VEGFR) therapies are used in cancer treatment, but their precise mechanisms are not fully understood.
- Adenovirus expressing VEGF-A (Ad-VEGF-A164) in mice creates tumor-like vasculature, including initial mother vessels (MV).
Purpose of the Study:
- To elucidate the rapid mechanisms of anti-VEGF/VEGFR drugs on tumor vasculature.
- To investigate the role of endothelial nitric oxide synthase (eNOS) in the action of anti-VEGF/VEGFR drugs.
- To characterize the formation and fate of glomeruloid microvascular proliferations (GMP) induced by these therapies.
Main Methods:
- Utilized an adenovirus expressing VEGF-A (Ad-VEGF-A164) model in mice to study tumor vasculature.
- Administered single doses of anti-VEGF/VEGFR drugs and quantified vascular changes using an Evans blue dye assay.
- Investigated the effect of an eNOS inhibitor (N(G)-Nitro-l-arginine methyl ester) on MV and GMP formation.
- Compared the effects of anti-VEGF drugs with the vasodisruptive drug combretastatin A4.
Main Results:
- A single dose of anti-VEGF/VEGFR drugs rapidly collapsed mother vessels (MV) into glomeruloid microvascular proliferations (GMP) within hours, with minimal endothelial cell death.
- GMP formation was identified as an intermediate step in the reversion of MV to normal microvessels.
- Inhibition of endothelial nitric oxide synthase (eNOS) mimicked the effects of anti-VEGF/VEGFR drugs, causing MV collapse to GMP.
- Ad-VEGF-A164 increased eNOS expression, and anti-VEGF/VEGFR drugs induced hypertension, consistent with eNOS inhibition and vasocontraction.
Conclusions:
- Anti-VEGF/VEGFR drugs primarily act by inhibiting eNOS, leading to vasocontraction and rapid collapse of MV into GMP.
- This process facilitates the subsequent normalization of tumor vasculature without causing widespread endothelial cell death.
- The findings clarify the mechanism of action for anti-VEGF therapies and highlight the role of eNOS in regulating tumor vascular responses.
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