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. 2018 Jan 1;28(1):31-43.
doi: 10.1089/ars.2017.7044. Epub 2017 Sep 21.

Flow-Responsive Vascular Endothelial Growth Factor Receptor-Protein Kinase C Isoform Epsilon Signaling Mediates Glycolytic Metabolites for Vascular Repair

Kyung In Baek  1 , Rongsong Li  2 , Nelson Jen  1 , Howard Choi  2 , Amir Kaboodrangi  1 , Peipei Ping  2   3 , David Liem  2 , Tyler Beebe  1 , Tzung K Hsiai  1   2   3   4   5
Affiliations

Flow-Responsive Vascular Endothelial Growth Factor Receptor-Protein Kinase C Isoform Epsilon Signaling Mediates Glycolytic Metabolites for Vascular Repair

Kyung In Baek et al. Antioxid Redox Signal. .

Abstract

Aims: Hemodynamic shear stress participates in maintaining vascular redox status. Elucidating flow-mediated endothelial metabolites enables us to discover metabolic biomarkers and therapeutic targets. We posited that flow-responsive vascular endothelial growth factor receptor (VEGFR)-protein kinase C isoform epsilon (PKCɛ)-6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) signaling modulates glycolytic metabolites for vascular repair.

Results: Bidirectional oscillatory flow (oscillatory shear stress [OSS]: 0.1 ± 3 dyne·cm-2 at 1 Hz) upregulated VEGFR-dependent PKCɛ expression to a greater degree than did unidirectional pulsatile flow (pulsatile shear stress [PSS]: 23 ± 8 dyne·cm-2 at 1 Hz) in human aortic endothelial cells (p < 0.05, n = 3). PSS and OSS further upregulated PKCɛ-dependent PFKFB3 expression for glycolysis (p < 0.05, n = 4). Constitutively active PKCɛ increased, whereas dominant-negative PKCɛ reduced both basal and maximal extracellular acidification rates for glycolytic flux (p < 0.01, n = 4). Metabolomic analysis demonstrated an increase in PKCɛ-dependent glycolytic metabolite, dihydroxyacetone (DHA), but a decrease in gluconeogenic metabolite, aspartic acid (p < 0.05 vs. control, n = 6). In a New Zealand White rabbit model, both PKCɛ and PFKFB3 immunostaining was prominent in the PSS- and OSS-exposed aortic arch and descending aorta. In a transgenic Tg(flk-1:EGFP) zebrafish model, GATA-1a morpholino oligonucleotide injection (to reduce viscosity-dependent shear stress) impaired vascular regeneration after tail amputation (p < 0.01, n = 20), which was restored with PKCɛ messenger RNA (mRNA) rescue (p < 0.05, n = 5). As a corollary, siPKCɛ inhibited tube formation and vascular repair, which were restored by DHA treatment in our Matrigel and zebrafish models. Innovation and Conclusion: Flow-sensitive VEGFR-PKCɛ-PFKFB3 signaling increases the glycolytic metabolite, dihydroxyacetone, to promote vascular repair. Antioxid. Redox Signal. 28, 31-43.

Keywords: PFKFB3; PKCɛ; dihydroxyacetone; metabolites; shear stress; vascular repair.

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Conflict of interest statement

No competing financial interests exist.

Figures

<b>FIG. 1.</b>
FIG. 1.
Shear stress-responsive VEGFR-PKCɛ-PFKFB3 signaling. (A) HAEC were transfected with scrambled (Scr) siRNA or VEGFR2 siRNA (siVEGFR2), or they were treated with or without VEGFR inhibitor. PSS and OSS differentially upregulated VEGFR2-dependent PKCɛ mRNA expression (*p < 0.05, n = 3). (B) PSS and OSS further upregulated PKCɛ activity (*p < 0.05, n = 3). (C) PSS and OSS also upregulated PFKFB3 mRNA expression, which was abrogated in response to siPKCɛ (*p < 0.05, n = 4). (D) CA-PKCɛ promoted, whereas DN-PKCɛ reduced the PFKFB3 mRNA expression (*p < 0.05 vs. LacZ control, n = 3). (E, F) Seahorse assay revealed that CA-PKCɛ increased both (E) basal and (F) max ECAR at the baseline and in response to H2O2 at 50 μM (*p < 0.01 vs. DN-PKCɛ, n = 4). CA, constitutively active; DN, dominant negative; ECAR, extracellular acidification rates; HAEC, human aortic endothelial cells; mRNA, messenger RNA; OSS, oscillatory shear stress; PFKFB3, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3; PKCɛ, protein kinase C isoform epsilon; PSS, pulsatile shear stress; siRNA, small interfering RNA; VEGFR, vascular endothelial growth factor receptor. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars
<b>FIG. 2.</b>
FIG. 2.
Shear stress-mediated PKCɛ-dependent metabolites. HAEC were transfected with scramble (Scr) siRNA or PKCɛ siRNA (siPKC), followed by three conditions: (i) static state, (ii) PSS, or (iii) OSS for 4 h. (A, C) Of the 136 known metabolites, PCA revealed significant overlapping of metabolites after the static, OSS, and PSS conditions in both Scr and siPKCɛ-transfected HAEC. (B, D) The concentration of 16 metabolites in Scr and 13 in siPKCɛ groups was significantly changed after the three conditions (*p < 0.05, n = 6). PCA revealed a separation among the statistically different metabolites. (E) PSS and OSS significantly modulated the selected metabolites, demonstrating PKCɛ-dependent Glucose, Frucose, DHA, and PKCɛ-independent Putrescine (*p < 0.05, n = 6). DHA, dihydroxyacetone; PCA, principal component analysis. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars
<b>FIG. 3.</b>
FIG. 3.
PKCɛ-mediated tube formation and vascular repair. (A) HAEC were transfected with scrambled (Scr) siRNA or PKCɛ siRNA (siPKCɛ). (B) siRNA attenuated tube formation, which was quantified by the changes in tube lengths. (C, D) HAEC were infected with recombinant control LacZ or CA-PKCɛ adenoviruses. Treatment with Cediranib at 10 μM inhibited tube formation (*p < 0.05, n = 5), whereas CA-PKCɛ restored Cediranib-attenuated tube formation (*p < 0.05, n = 5). (E) Transgenic Tg(flk-1:EGFP) embryos underwent tail amputation as a mode of vascular regeneration. The control group and p53 MO-injected embryos exhibited vascular repair (yellow arrows) by re-connecting DLAV with DA. PKCɛ MO injection impaired vascular repair (red arrow), whereas co-injecting PKCɛ mRNA restored vascular repair (yellow arrow). DA, dorsal aorta; DLAV, dorsal longitudinal anastomotic vessel; ISV, intersegmental vessel; MO, morpholino oligonucleotide; PCV, posterior cardinal vein; SIV, subintestinal vessel. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars
<b>FIG. 4.</b>
FIG. 4.
Shear stress-mediated vascular repair. (A, B) The control and p53 MO-injected fish developed vascular repair at 3 dpa (yellow arrows). (C) GATA-1a MO delayed vascular repair at 3 dpa. (D) TNNT-2 MO impaired vascular repair (red arrow) at 3 dpa (*p < 0.05, n = 5), and the embryos failed to thrive at 5 dpa. (E) EPO mRNA injection promoted vascular repair at 3 dpa. (F) Co-injection of GATA-1a MO with PKCɛ mRNA resulted in vascular repair. (G) Quantitative comparison revealed differential percentage of vascular repair (*p < 0.05, n = 30; n = 5 for TNNT-2 MO injected group). dpa, days post-amputation; EPO, erythropoietin. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars
<b>FIG. 5.</b>
FIG. 5.
Glycolytic metabolite DHA promoted vascular repair. (A–B) DHA promoted tube formation in HAEC transfected with scrambled (Scr) siRNA. siPKCɛ attenuated tube formation, which was rescued with DHA at 1 mg/ml (*p < 0.05, n = 4). (B) Provides quantifications of the relative tube length for (A). (C) Tg (flk-1:EGFP) embryos injected with p53 MO or PKCɛ MO underwent tail amputation, and they were treated with or without DHA (1 mg/ml) for 3 days. DHA treatment rescued impaired vascular repair after PKCɛ MO injection. (D) Vascular repair was quantified by the percentage of embryos that developed vascular regeneration (n = 20). To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars
<b>FIG. 6.</b>
FIG. 6.
A schematic diagram depicts flow-sensitive VEGFR-PKCɛ-PFKFB3 modulation of glycolytic metabolites for vascular repair. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars
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