Vascular endothelial growth factor (VEGF) exerts crucial functions during pathological angiogenesis and normal physiology. We observed increased hematocrit (60–75%) after high-grade inhibition of VEGF by diverse methods, including adenoviral expression of soluble VEGF receptor (VEGFR) ectodomains, recombinant VEGF Trap protein and the VEGFR2-selective antibody DC101. Increased production of red blood cells (erythrocytosis) occurred in both mouse and primate models, and was associated with near-complete neutralization of VEGF corneal micropocket angiogenesis. High-grade inhibition of VEGF induced hepatic synthesis of erythropoietin (Epo, encoded by Epo) >40-fold through a HIF-1α–independent mechanism, in parallel with suppression of renal Epo mRNA. Studies using hepatocyte-specific deletion of the Vegfa gene and hepatocyte–endothelial cell cocultures indicated that blockade of VEGF induced hepatic Epo by interfering with homeostatic VEGFR2-dependent paracrine signaling involving interactions between hepatocytes and endothelial cells. These data indicate that VEGF is a previously unsuspected negative regulator of hepatic Epo synthesis and erythropoiesis and suggest that levels of Epo and erythrocytosis could represent noninvasive surrogate markers for stringent blockade of VEGF in vivo.
NOTE: In the version of this article initially published, the name of one of the authors, Nihar R. Nayak, was misspelled as Nihar R. Niyak. The error has been corrected in the HTML and PDF versions of the article.
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We thank members of the Kuo laboratory for discussion, J. Folkman for support of this project, C. Davis for histopathologic analysis of liver sections, C.-P. Chang for HA-Flt1-His cDNA, A. Patterson for determination of arterial oxygen tension, A. Wagers for demonstrating parabiotic surgeries, M. Socolovsky for murine Epo, J. Zehnder for advice with real-time PCR, C. Koch for EF5 and EF5-specific antibodies, G. Molineux for permission to cite unpublished data and E. Yu for initial assistance. We are indebted to H.-P. Gerber and N. Ferrara for their gift of VegfaloxP/loxP mice, Y.-M. Lin and T. Wandless for synthesis of ZD4190 and the UCSF Liver Center (supported by NIH P30 DK26743) for providing core services. B.Y.Y.T. is a Fonds de la Recherche en Santé du Québec fellow. K.W. is supported by a Medical Scientist Training Program Grant to Stanford University. This work was supported by grants from the US National Institutes of Health (1 R01 CA95654-01 and 1 R01 HL074267-01) and the Department of Defense (DAMD17-02-1-0143) to C.J.K. and funds from Amgen Corporation (to R.J.K.). C.J.K. is a Burroughs Wellcome Foundation Scholar in the Pharmacological Sciences and a Kimmel Foundation Scholar.
The authors declare no competing financial interests.
Elevation of erythroid parameters in sVEGFR-treated mice (PDF 139 kb)
Stimulation of Epo mRNA production in hepatocytes of Ad-Flt1treated mice as determined by in situ hybridization. (PDF 519 kb)
Soluble Flt1 ectodomain induces hepatic Epo expression in the absence of direct liver infection in parabiotic mice. (PDF 117 kb)
Transient increase of Epo mRNA in hypoxic kidneys or livers. (PDF 60 kb)
Ad-Cremediated excision of the loxP-flanked stop cassette in ROSA26-LacZ mice allows expression of the lacZ gene. (PDF 2327 kb)
EF5 detection of hypoxia in tumors and Ad-Flt1treated liver. (PDF 206 kb)
Ad-Flt1 and Ad-Flk1-Fctreated livers do not show evidence of hemorrhage, necrosis or ischemic tissue damage. (PDF 818 kb)
Epo mRNA is selectively elevated in hepatocyte coculture with LSECs but not other endothelial cell types or fibroblasts. (PDF 117 kb)
Comparative inhibition of VEGF-stimulated corneal-micropocket angiogenesis by Ad-Flt1, ZD4190 and rVEGF Trap. (PDF 490 kb)
Elevation of Ter119+CD45 erythroid precursors by VEGF blockade. (PDF 58 kb)
Assessment of hydration and oxygenation status following VEGF inhibition. (PDF 117 kb)
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