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.


Available from: Aleksander S Popel
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    • "In some circumstances, excessive influx of VEGF-A (either exogenous for cells in vitro, or endogenous for ischemia models in vivo) may dysregulate intrinsic VEGFR balance of target cells and switch them to VEGFR1 expression or may upregulate soluble VEGFR1 expression that can operate as a negative feedback system, thereby undermining the entire positive effect of the treatment [22][23][24]. Moreover, the qualitative and quantitative balance of VEGFR1 and VEGFR2 proteins may vary in human populations, further complicating the proper choice of treatment for some cases [23]. Considering this, any VEGF-A-based pro-angiogenic therapy may prove to be more reliable and lead to more predictable results when supported by some additional VEGF-A-independent line of intervention (e.g., using UC-MSCs). "
    [Show abstract] [Hide abstract] ABSTRACT: Mesenchymal stromal/stem cells derived from human umbilical cord (UC-MSCs) uniquely combine properties of embryonic and postnatal MSCs and may be the most acceptable, safe, and effective source for allogeneic cell therapy e.g. for therapeutic angiogenesis. In this report we describe pro-angiogenic properties of UC-MSCs as manifested in vitro. UC-MSCs were isolated from human Wharton’s jelly by enzymatic digestion. Presence of soluble forms of VEGF-A in UC-MSC-conditioned media was measured by ELISA. Effects of the conditioned media on human umbilical vein-derived endothelial EA.hy926 cells proliferation were measured by MTT-assay; changes in cell motility and directed migration were assessed by scratch wound healing and transwell chamber migration assays. Angiogenesis was modeled in vitro as tube formation on basement membrane matrix. Progressive differentiation of MSCs to endothelioid progeny was assessed by CD31 immunostaining. Although no detectable quantities of soluble VEGF-A were produced by UC-MSCs, the culture medium, conditioned by the UC-MSCs, effectively stimulated proliferation, motility, and directed migration of EA.hy926 cells. In 2D culture, UC-MSCs were able to acquire CD31 + endothelial cell-like phenotype when stimulated by EA.hy926-conditioned media supplemented with VEGF-A165. UC-MSCs were capable of forming unstable 2D tubular networks either by themselves or in combinations with EA.hy926 cells. Active spontaneous sprouting from cell clusters, resulting from disassembling of such networks, was observed only in the mixed cultures, not in pure UC-MSC cultures. In 3D mode of sprouting experimentation, structural support of newly formed capillary-like structures was provided by UC-MSCs that acquired the CD31 + phenotype in the absence of exogenous VEGF-A. These data suggest that a VEGF-A-independent paracrine mechanism and at least partially VEGF-A-independent differentiation mechanism are involved in the pro-angiogenic activity of UC-MSCs.
    Full-text · Article · Dec 2016 · Stem Cell Research & Therapy
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    • "Several researchers believe that endothelial progenitor cells included in BMCs differentiated into endothelial cells and, after transplantation in the injury region, contributes to cell survival and angiogenesis[17,18]. Autologous BMCs and PRP are a rich source of growth factors that stimulates angiogenesis in ischemic muscle[19]. Moreover, angiogenic factors, including VEGF and epithelial growth factor (EGF), are also released from injured tissue recruiting BMCs to replace injured tissue[20,21].Previous studies similarly demonstrated that there is an additional contribution derived from BMCs or PRP progenitors which are mobilized in the setting of limb or myocardial ischemia, migrate to ischemic tissue, and are actively incorporate into new vessels[22]. "
    Full-text · Article · Jan 2015 · Journal of Clinical and Experimental Cardiology
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    • "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.
    Full-text · Article · Sep 2014 · OncoTargets and Therapy
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