Distribution of vascular endothelial cell growth factor A (VEGF-A) as a gradient determines microvascular endothelial cell (EC) fate during organogenesis. While much is understood about mechanisms of differential distribution, less is known about how EC perceive and interpret a graded VEGF-A signal to generate positional target gene activation. Using microvascular EC, we analyzed the effect of time and graded VEGF-A input on VEGFR2 autophosphorylation, signal kinase activation and induction of immediate-early genes. The threshold and time to peak activation of VEGFR2 were dependent on signal strength over a 50-fold range in concentration with 3-fold concentration differences readily distinguished. Longer duration of exposure did not compensate for low concentration of VEGF-A, suggesting intensity and duration of signal were not interpreted equivalently. With the same conditions, graded and time-sensitive information was transduced through the PLCgamma/p44/p42MAPK signal pathway but not the parallel AKT pathway. Analysis of MAPK-induced angiogenic immediate-early genes determined that EGR-1, EGR-3, and NR4A1 were dependent on graded input while NR4A2 and DSCR1 were independent with 'switch-like' induction. These data demonstrate rapid, linear integration of VEGF-A levels but independent interpretation of duration of signal and identify potential nodes for segregation of gradient-dependent and -independent responses. These results describe how microvascular EC fate decisions can be determined by comparatively moderate changes in VEGF signal strength, resulting in combinatorial changes in the repertoire of immediate-early genes for transcription effectors.
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"We hypothesize that in the iPSC-EC sprouting experiments, the N-VBP microspheres initially sequestered VEGF and served as a 'source' for local VEGF release (Fig. 5A). N-VBP microspheres encapsulated in three dimensional hydrogels could also have contributed to the formation of spatial VEGF gradients within the hydrogels , which would be expected to promote iPSC-EC chemotaxis and sprouting [49,50]. Future work will be needed to examine this hypothesis by pre-incubating VBP microspheres in VEGF-containing medium and subsequently encapsulating the VEGF-containing microspheres in the sprouting assay to generate a uniform spatial distribution of VEGF throughout the hydrogel. "
[Show abstract][Hide abstract]ABSTRACT: Vascular endothelial growth factor (VEGF) spatial and temporal activity must be tightly controlled during angiogenesis to form perfusable vasculature in a healing wound. The native extracellular matrix (ECM) regulates growth factor activity locally via sequestering, and researchers have used ECM-mimicking approaches to regulate the activity of VEGF in cell culture and in vivo. However, the impact of dynamic, affinity-mediated growth factor sequestering has not been explored in detail with biomaterials. Here, we sought to modulate VEGF activity dynamically over time using poly(ethylene glycol) microspheres containing VEGF-binding peptides (VBPs) and exhibiting varying degradation rates. The degradation rate of VBP microspheres conferred a differential ability to up- or down-regulate VEGF activity in culture with primary human endothelial cells. VBP microspheres with fast-degrading crosslinks reduced VEGF activity and signaling, while VBP microspheres with no inherent degradability sequestered and promoted VEGF activity in culture with endothelial cells. VBP microspheres with degradable crosslinks significantly reduced neovascularization in vivo, but neither non-degradable VBP microspheres nor bolus delivery of soluble VBP reduced neovascularization. The covalent incorporation of VBP to degradable microspheres was required to reduce neovascularization in a mouse model of choroidal neovascularization in vivo, which demonstrates a potential clinical application of degradable VBP microspheres to reduce pathological angiogenesis. The results herein highlight the ability to modulate the activity of a sequestered growth factor by changing the crosslinker identity within PEG hydrogel microspheres. The insights gained here may instruct the design and translation of affinity-based growth factor sequestering biomaterials for regenerative medicine applications.
"For instance, the differential activation of jagged1 and DII4 in the Notch signaling pathway determines the distinct fate (tip vs. stalk) of two adjacent endothelial cells exposed to a VEGF gradient during the angiogenic process . A recent study has further demonstrated that the mitogen-activated protein kinase pathway is triggered by VEGF in individual microvascular endothelial cells, whereas their adjoining cells are silent . The dissimilar response of neighboring cells to VEGF might be linked to the wide variation in VEGFR-2 levels, as only about 60% of UCB-ECFCs express it (unpublished data from our group). "
[Show abstract][Hide abstract]ABSTRACT: Endothelial colony forming cells (ECFCs) are the only endothelial progenitor cells (EPCs) capable of acquiring a mature endothelial phenotype. ECFCs are mainly mobilized from bone-marrow to promote vascularization and represent a promising tool for cell-based therapy of severe ischemic diseases. VEGF stimulates the proliferation of peripheral blood-derived ECFCs (PB-ECFCs) through oscillations in intracellular Ca2+ concentration ([Ca2+]i). VEGF-induced Ca2+ spikes are driven by the interplay between inositol-1,4,5-trisphosphate (InsP3)-dependent Ca2+ release and store-operated Ca2+ entry (SOCE). The therapeutic potential of umbilical cord blood-derived ECFCs (UCB-ECFCs) has also been shown in recent studies. However, VEGF-induced proliferation of UCB-ECFCs is faster compared to their peripheral counterpart. Unlike PB-ECFCs, UCB-ECFCs express canonical transient receptor potential channel 3 (TRPC3), that mediates diacylglycerol-dependent Ca2+ entry. The present study aimed to investigate whether the higher proliferative potential of UCB-ECFCs was associated to any difference in the molecular underpinnings of their Ca2+ response to VEGF. We found that VEGF induces oscillations in [Ca2+]i that are patterned by the interaction between InsP3-dependent Ca2+ release and SOCE. Unlike PB-ECFCs, VEGF-evoked Ca2+ oscillations do not arise in the absence of extracellular Ca2+ entry and after pharmacological (with Pyr3 and flufenamic acid) and genetic (by employing selective small interference RNA) suppression of TRPC3. VEGF-induced UCB-ECFC proliferation is abrogated upon inhibition of the intracellular Ca2+ spikes. Therefore, the Ca2+ response to VEGF in UCB-ECFCs is shaped by a different Ca2+ machinery as compared to PB-ECFCs and TRPC3 stands out as a promising target in EPC-based treatment of ischemic pathologies.
Full-text · Article · May 2013 · Stem cells and development
"VEGFA and EPO stimulate angiogenesis, and EPO additionally enhances the production of red blood cells. Endothelial cells are the predominant target of VEGFA, because of their ability to grow towards a hypoxic region following a VEGFA gradient and thereby forming new blood vessels [4,5]. The cellular signaling response to hypoxia needs to be tightly regulated, since already a small deviation of the balance between pro-and anti-angiogenic factors can cause several severe diseases such as ocular and inflammatory disorders. "
[Show abstract][Hide abstract]ABSTRACT: Adaptation to low oxygen by changing gene expression is vitally important for cell survival and tissue development. The sprouting of new blood vessels, initiated from endothelial cells, restores the oxygen supply of ischemic tissues. In contrast to the transcriptional response induced by hypoxia, which is mainly mediated by members of the HIF family, there are only few studies investigating alternative splicing events. Therefore, we performed an exon array for the genome-wide analysis of hypoxia-related changes of alternative splicing in endothelial cells.
Human umbilical vein endothelial cells (HUVECs) were incubated under hypoxic conditions (1% O(2)) for 48 h. Genome-wide transcript and exon expression levels were assessed using the Affymetrix GeneChip Human Exon 1.0 ST Array. We found altered expression of 294 genes after hypoxia treatment. Upregulated genes are highly enriched in glucose metabolism and angiogenesis related processes, whereas downregulated genes are mainly connected to cell cycle and DNA repair. Thus, gene expression patterns recapitulate known adaptations to low oxygen supply. Alternative splicing events, until now not related to hypoxia, are shown for nine genes: six which are implicated in angiogenesis-mediated cytoskeleton remodeling (cask, itsn1, larp6, sptan1, tpm1 and robo1); one, which is involved in the synthesis of membrane-anchors (pign) and two universal regulators of gene expression (cugbp1 and max).
For the first time, this study investigates changes in splicing in the physiological response to hypoxia on a genome-wide scale. Nine alternative splicing events, until now not related to hypoxia, are reported, considerably expanding the information on splicing changes due to low oxygen supply. Therefore, this study provides further knowledge on hypoxia induced gene expression changes and presents new starting points to study the hypoxia adaptation of endothelial cells.