Endothelial cell-by-cell profiling reveals the temporal dynamics of VEGFR1 and VEGFR2 membrane localization after murine hindlimb ischemia

1University of Illinois.
AJP Heart and Circulatory Physiology (Impact Factor: 3.84). 02/2013; 304(8). DOI: 10.1152/ajpheart.00514.2012
Source: PubMed


Vascular endothelial factor receptor (VEGFR) cell surface localization plays a critical role in transducing VEGF signaling towards angiogenic outcomes and quantitative characterization of these parameters is critical to advancing computational models for predictive medicine. However data to this point has largely examined intact muscle, thus essential data on the cellular localization of the receptors within the tissue are currently unknown. Therefore, our aims are to quantitatively analyze VEGFR localization on endothelial cells from mouse hindlimb skeletal muscles following the induction of hindlimb ischemia, an established model for human peripheral artery disease. Flow cytometry is used to measure and compare the ex vivo surface localization of VEGFR1 and VEGFR2 on CD31(+)/CD34(+) endothelial cells, 3 and 10 days after unilateral ligation of the femoral artery. We determine that 3 days after hindlimb ischemia VEGFR2 surface-levels are decreased by 80% compared to endothelial cells from the non-ischemic limb, and 10 days after ischemia, we observe a 2-fold increase in the surface-levels of the modulatory receptor, VEGFR1, along with increased PCNA, uPA, and uPAR mRNA expression, compared to the non-ischemic limb. The significant upregulation of VEGFR1 surface-levels indicates that VEGFR1 indeed plays a critical role in the ischemia-induced perfusion-recovery process, a process that includes both angiogenesis and arteriogenesis. The quantification of these dissimilarities for the first time, ex vivo, provides insight into the balance of modulatory (VEGFR1) and pro-angiogenic (VEGFR2) receptors in ischemia and lays a foundation for systems biology approaches towards therapeutic angiogenesis.

Download full-text


Available from: Aleksander S Popel, Oct 05, 2015
31 Reads
  • Source
    • "Previous studies have been effective at measuring the numbers of VEGFRs in vitro29 and in vivo.30,31 Although the changes in VEGFRs have been investigated under ischemia,32 to our knowledge, the changes in surface receptors after antiangiogenic treatment have not been studied. Therefore, we investigate the changes in the number of VEGFR2 molecules on the endothelial cell surface after antiangiogenic treatment to better understand the cellular response to treatment. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Angiogenesis, the formation of new blood vessels, is an essential step for cancer progression, but antiangiogenic therapies have shown limited success. Therefore, a better understanding of the effects of antiangiogenic treatments on endothelial cells is necessary. In this study, we evaluate the changes in cell surface vascular endothelial growth factor receptor (VEGFR) expression on endothelial cells in culture treated with the antiangiogenic tyrosine kinase inhibitor drug sunitinib, using quantitative flow cytometry. We find that proangiogenic VEGFR2 cell surface receptor numbers are increased with sunitinib treatment. This proangiogenic effect might account for the limited effects of sunitinib as a cancer therapy. We also find that this increase is inhibited by brefeldin A, an inhibitor of protein transport from the endoplasmic reticulum to the Golgi apparatus. The complex dynamics of cell surface VEGFRs may be important for successful treatment of cancer with antiangiogenic therapeutics.
    OncoTargets and Therapy 09/2014; 7:1571-82. DOI:10.2147/OTT.S65055 · 2.31 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: How single chain urokinase (ScuPA) mediates angiogenesis is incompletely understood. ScuPA (>4 nM) induces pERK1/2 (MAPK44 and 42) and pAkt (S(473)) in umbilical vein and dermal microvascular endothelial cells. Activation of pERK1/2 by ScuPA is blocked by PD98059 or U0126 and pAktS(473) activation is inhibited by Wortmannin or LY294002. ScuPA (32 nM) or protease-inhibited two-chain urokinase stimulates pERK1/2 to the same extent, indicating that signaling is not dependent on enzymatic activity. ScuPA induces pERK1/2, but not pAktS(473), in SIN1(-/-) cells indicating that the two pathways are not identical. Peptides from domain 2 of the urokinase plasminogen activator receptor (uPAR) or domain 5 of high molecular weight kininogen (HK) compete with ScuPA for induction of pERK1/2 and pAktS473. A peptide to the integrin binding site on uPAR, a β1 integrin peptide that binds uPAR, antibody 6S6 to β1 integrin, the tyrosine kinase inhibitors AG1478 or PP3, and siRNA knockdown of VEGFR2, but not HER1-4, blocks ScuPA-induced pERK1/2 and pAktS(473). ScuPA-induced endothelial cell proliferation is blocked by inhibitors of pERK1/2 and pAktS(473), antibody 6S6, and uPAR or kininogen peptides. ScuPA initiates aortic sprouts and matrigel plug angiogenesis in normal, but not uPAR deficient, mouse aortae or mice, respectively, but they are blocked by PD98059, LY294002, AG1478 or cleaved HK. In sum, this investigation indicates a novel, a non-proteolytic signaling pathway initiated by zymogen ScuPA and mediated by domain 2 of uPAR, β1 integrins, and VEGFR2 leading to angiogenesis. Kininogens or peptides from it down regulate this pathway.
    AJP Heart and Circulatory Physiology 05/2013; 305(3). DOI:10.1152/ajpheart.00110.2013 · 3.84 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Plasma membrane-localized vascular endothelial growth factor receptors (VEGFR) play a critical role in transducing VEGF signaling toward pro and antiangiogenic outcomes and quantitative characterization of these receptors is critical toward identifying biomarkers for antiangiogenic therapies, understanding mechanisms of action of antiangiogenic drugs, and advancing predictive computational models. While in vitro analysis of cell surface-VEGFRs has been performed, little is known about the levels of cell surface-VEGFR on tumor cells. Therefore, we inoculate nude mice with the human triple-negative breast cancer, MDA-MB-231, cell line; isolate human tumor cells and mouse tumor endothelial cells from xenografts; and quantitatively characterize the VEGFR localization on these cells. We observe 15,000 surface-VEGFR1/tumor endothelial cell versus 8200 surface-VEGFR1/tumor endothelial cell at 3 and 6 weeks of tumor growth, respectively; and we quantify 1200-1700 surface-VEGFR2/tumor endothelial cell. The tumor cell levels of VEGFR1 and VEGFR2 are relatively constant between 3 and 6 weeks: 2000-2200 surface-VEGFR1/tumor cell and ~1000 surface-VEGFR2/tumor cell. Cell-by-cell analysis provides additional insight into tumor heterogeneity by identifying four cellular subpopulations based on size and levels of cell membrane-localized VEGFR. Furthermore, when these ex vivo data are compared to in vitro data, we observe little to no VEGFRs on MDA-MB-231 cells, and the MDA-MB-231 VEGFR surface levels are not regulated by a saturating dose of VEGF. Overall, the quantification of these dissimilarities for the first time in tumor provides insight into the balance of modulatory (VEGFR1) and proangiogenic (VEGFR2) receptors.
    Cancer Medicine 04/2014; 3(2). DOI:10.1002/cam4.188 · 2.50 Impact Factor
Show more