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

Abstract

Imoukhuede PI, Dokun AO, Annex BH, Popel AS. Endothelial cell-by-cell profiling reveals the temporal dynamics of VEGFR1 and VEGFR2 membrane localization after murine hindlimb ischemia. Am J Physiol Heart Circ Physiol 304: H1085-H1093, 2013. First published February 1, 2013 doi:10.1152/ajpheart.00514.2012 cell surface localization plays a critical role in transducing VEGF signaling toward angiogenic outcomes, and quantitative characterization of these parameters is critical to advancing computational models for predictive medicine. However, studies to this point have largely examined intact muscle; thus, essential data on the cellular localization of the receptors within the tissue are currently unknown. Therefore, our aims were to quantitatively analyze VEGFR localization on endothelial cells (ECs) from mouse hindlimb skeletal muscles after the induction of hindlimb ischemia, an established model for human peripheral artery disease. Flow cytometry was used to measure and compare the ex vivo surface localization of VEGFR1 and VEGFR2 on CD31 ϩ /CD34 ϩ ECs 3 and 10 days after unilateral ligation of the femoral artery. We determined that 3 days after hindlimb ischemia, VEGFR2 surface levels were decreased by 80% compared with ECs from the nonischemic limb; 10 days after ischemia, we observed a twofold increase in surface levels of the modulatory receptor, VEGFR1, along with increased proliferating cell nuclear antigen, urokinase plasminogen activator, and urokinase plasminogen activator receptor mRNA expression compared with the nonischemic 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 insights into the balance of modulatory (VEGFR1) and proangiogenic (VEGFR2) receptors in ischemia and lays the foundation for systems biology approaches toward therapeutic angiogenesis.angiogenesis; peripheral artery disease; endothelial cells; skeletal muscle; quantitative flow cytometry; receptor localization; Quantibrite; hindlimb ischemia; vascular endothelial growth factor receptor THE VEGF RECEPTOR (VEGFR)-ligand family is a key regulator of angiogenesis, the growth of new blood vessels from preexisting microvasculature. VEGF binding to its receptors activates intracellular signaling pathways inducing the endothelial proliferation and migration necessary for angiogenesis. Therapeutic angiogenesis aims to treat ischemic disease through the supplementation of VEGF and/or other angiogenic growth factors (31,48). The use of VEGF has successfully stimulated neovascularization in animal models of ischemia (30, 68). However, the use of VEGFs, fibroblast growth factor, hepatocyte growth factor, and other proangiogenic molecules has not yielded successful clinical outcomes (8,31,34). These clinical setbacks have been attributed to inadequate dosing, duration, and delivery as well a...

Activator of G-protein signaling 8 is involved in VEGF-mediated signal processing during angiogenesis

“…The counterbalanced expression of VEGFR-1 and VEGFR-2 in endothelial cells has been reported during hypoxia (Marti and Risau, 1998;Ulyatt et al, 2011), ischemia (Imoukhuede et al, 2013) and VEGF treatment (Imoukhuede and Popel, 2011). VEGFR-1 is an isoform with higher VEGF affinity but less kinase activity than VEGFR-2, suggesting a decoy function for VEGFR-1 by sequestering VEGF (Roskoski, 2008).…”

ADAM12: a genetic modifier of preclinical peripheral arterial disease

“…In vitro simulation of ischemia was achieved as described previously (30) with modifications as follows: cells were deprived of nutrients and oxygen by growing in endothelial starvation medium (Cell Applications) and cultured in 2% oxygen in a hypoxic chamber fitted with a ProOx model 110 oxygen controller for continuous monitoring and adjustment of the oxygen content of the chamber (BioSpherix, Lacona, NY). In experiments using CD31 ϩ cells from mouse hindlimbs, the cells were isolated, as described previously (19), and detailed below.…”

“…Cells were isolated from mouse hindlimbs, as described previously (19). CD31-expressing cells were isolated from mouse hindlimbs, as described previously (19). Briefly, GA and TA muscles were extracted from mouse hindlimbs.…”

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“…It is notable that VEGFR2 was recently shown to be downregulated in myocardium by I/R injury 39 or in hindlimb muscles by acute ischemia 40,41 in rodents. It is generally recognized that most biological functions of VEGF are mediated via VEGFR2, one of the two receptor protein tyrosine kinases, VEGFR1 (Flt-1) and VEGFR2 (KDR), to which VEGF binds.…”