Erythropolesis, the production of red blood cells (RBCs), is regulated by an intricate network of signals involving the hormone erythropoietin (Epo), and sensing of hypoxia through inhibition of both HIF-1alpha proline hydroxylation and its subsequent interaction with the Von Hippel-Lindau (VHL) protein. Either insufficient or excess erythropoiesis produces disease in humans, with too few RBCs resulting in anemia, and too many RBCs resulting in polycythemia vera. The treatment of anemia alone consumes significant economic resources, with the necessity for either blood transfusions or Epo treatment. We have developed adenoviruses which express soluble ectodomains of the Flkl and Fltl receptors for Vascular Endothelial Growth Factor (VEGF). Single injections in mice of these adenoviruses produce high levels of circulating soluble Flk1 and Flt1 ectodomains which persist for >3-4 weeks, potently suppressing tumor growth and angiogenesis, and producing stringent conditional inactivation of-VEGF in adult animals. Surprisingly, adenoviral expression of Flk1 or Flt1 ectodomains (soluble VEGF receptors, or sVEGFRs) in normal mice strongly stimulates erythropoiesis, with increase in hematocrit increasing from baseline 45% to new levels of 55-75 %. Circulating erythropoietin levels are elevated in parallel; however, the Epo originates from the liver as in embryonic development, not the usual site of synthesis in the kidney. This data implicates VEGF as an unsuspected repressor of hepatic Epo synthesis, and this grant application is focused on determining the underlying moIecular mechanisms.
In Aim 1, experiments will expand upon preliminary data implicating perturbation of hepatocyte--endothelial cross-talk in the sVEGFR induction of hepatic Epo. Adenoviral and transgenic expression of Cre recombinase will be used to test the effects of specific inhibition of hepatocyte-produced VEGF on hepatocyte Epo expression. Conversely, co-culture experiments manipulating VEGF will be used to test the effects of endothelial cells on hepatocyte Epo production.
Aim 2 will define the specific factors mediating the transcriptional regulation of Epo by VEGF and VEGF blockade. Candidate factors (HIF proteins, HNF4, RXRalpha, GATA factors) will be directly tested by chromatin immunoprecipitation assays and functionally by adenoviral and transgenic expression of Cre to delete floxed alleles of relevant candidates. Additionally, novel factors will be sought by unbiased approaches of DNAse1 footprinting and hypersensitivity assays. The therapeutic implications of the ability to reactivate hepatic Epo synthesis are substantial. These studies should also yield significant insight into the action of VEGF as an unsuspected upstream regulator of RBC homeostasis, into the regulation of hepatic Epo production which is strongly repressed post-natally, and indicate the potential utility of alterations in erythropoiesis as surrogate markers for or a desirable consequence of stringent VEGF blockade.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL074267-02
Application #
7093603
Study Section
Hematopoiesis Study Section (HP)
Program Officer
Qasba, Pankaj
Project Start
2005-07-15
Project End
2010-05-31
Budget Start
2006-06-01
Budget End
2007-05-31
Support Year
2
Fiscal Year
2006
Total Cost
$346,122
Indirect Cost
Name
Stanford University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
Chang, Junlei; Mancuso, Michael R; Maier, Carolina et al. (2017) Gpr124 is essential for blood-brain barrier integrity in central nervous system disease. Nat Med 23:450-460
Wei, Kevin; Piecewicz, Stephanie M; McGinnis, Lisa M et al. (2013) A liver Hif-2?-Irs2 pathway sensitizes hepatic insulin signaling and is modulated by Vegf inhibition. Nat Med 19:1331-1337
Taniguchi, Cullen M; Finger, Elizabeth C; Krieg, Adam J et al. (2013) Cross-talk between hypoxia and insulin signaling through Phd3 regulates hepatic glucose and lipid metabolism and ameliorates diabetes. Nat Med 19:1325-30
Riess, Jonathan W; Logan, Aaron C; Krupitskaya, Yelena et al. (2012) Maintenance bevacizumab is associated with increased hemoglobin in patients with advanced, nonsquamous, non-small cell lung cancer. Cancer Invest 30:231-5
Lin, Shuei-Liong; Chang, Fan-Chi; Schrimpf, Claudia et al. (2011) Targeting endothelium-pericyte cross talk by inhibiting VEGF receptor signaling attenuates kidney microvascular rarefaction and fibrosis. Am J Pathol 178:911-23
Fuxe, Jonas; Lashnits, Erin; O'Brien, Shaun et al. (2010) Angiopoietin/Tie2 signaling transforms capillaries into venules primed for leukocyte trafficking in airway inflammation. Am J Pathol 176:2009-18
Stankunas, Kryn; Ma, Gene K; Kuhnert, Frank J et al. (2010) VEGF signaling has distinct spatiotemporal roles during heart valve development. Dev Biol 347:325-36
Kuhnert, Frank; Mancuso, Michael R; Shamloo, Amir et al. (2010) Essential regulation of CNS angiogenesis by the orphan G protein-coupled receptor GPR124. Science 330:985-9
Harshman, Lauren C; Kuo, Calvin J; Wong, Bryan Y et al. (2009) Increased hemoglobin associated with VEGF inhibitors in advanced renal cell carcinoma. Cancer Invest 27:851-6
Chan, Charles K F; Chen, Ching-Cheng; Luppen, Cynthia A et al. (2009) Endochondral ossification is required for haematopoietic stem-cell niche formation. Nature 457:490-4

Showing the most recent 10 out of 15 publications