Endothelial TrkA coordinates vascularization and innervation in thermogenic adipose tissue and can be targeted to control metabolism

Figures

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  Figure 1. Endothelial TrkA isoform is the target of PEP3 in AT. (A) PEP3 receptor isolation from mouse BAT membrane protein extract. Upon elution with PEP3 or negative control peptide (WAT7), the specific band in silver-stained SDS-PAGE gel (arrow) was identified as TrkA. (B) Alignment of mouse NGF amino acid stretch 173–189 and PEP3. Red: residues in contact with TrkA. Lines: identity; dots: amino acids of similar class. (C) HUVEC sprouting promoted by NGF (50 nM) and blocked by 50 nM of PEP3. (D) Data from C quantified; Error bars: SEM, ∗P < 0.01 (Student's t-test); N = 5 spheroids. (E) PEP3 (biotinylated) binding to EC (endomucin+ IF) in SVF from SAT is blocked by equimolar concentration (1 ug/ml) of NGF. Yellow arrow: PEP3-bound EC. Blue arrow: non-endothelial cell (not PEP3-bound. (F) PEP3 receptor isolation performed from extracts of WT vs TrkA-EC-KO (KO) mice. Immunoblotting with anti-TrkA antibodies reveals the 70 kDa (arrow) and a smaller (arrowhead) isoforms missing in KO. NS: non-specific bands. (G) After 2 × 109 transforming units (TU) of phage displaying PEP3 injected into mice for 1 h, PEP3-phage accumulates more in BAT and SAT of WT mice compared to KO littermates; there is…

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  Figure 3. D-BAT induces apoptosis selectively in EC of thermogenic AT. (A) A model predicting D-BAT effect in AT. D-BAT depletes EC expressing the PEP3-tsargeted TrkA isoform while sparing SNS. Reduced BAT and SAT vascularization results in AT whitening and reduced thermogenesis. (B–F) C57BL/6 males were treated with D-BAT or PBS (control) over 2 weeks (N = 5). (B) Core body temperature maintenance at 4 °C measured 3 days after the last injection indicating reduced cold tolerance upon D-BAT treatment. Plotted are mean ± SEM; ∗P < 0.05 (Student's t-test). (C) Interscapular BAT 3 days after the last injection of D-BAT. (D) IF analysis of paraffin tissue sections from D-BAT-treated and control mice with cleaved caspase 3 IF (green) co-localized with IB4 (red). Note apoptosis in both endothelium (yellow arrow) and adipocytes (green arrow) of D-BAT-treated mice. (E) BAT IF for UCP1 (green)/perilipin1 (PLN1). Graph indicates decreased frequency of UCP1+ adipocytes, which are partly replaced by white adipocytes in D-BAT-treated mice. (F) Representative images of H/E-stained paraffin sections of BAT and SAT from treated and control mice with quantifications to the right. Arrow: beige adipocyte…

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  Figure 4. D-BAT attenuates tumor-associated AT browning and lipolysis. C57BL/6 female mice (N = 5), pre-treated with D-BAT or PBS (control) over 3 weeks, were grafted with tumors and then injected with D-BAT or PBS for 2 more weeks. (A) BAT, tumor-distal SAT, tumor-proximal VAT and tumors from representative mice. (B) Quantification of tissue mass from all mice (N = 5). Plotted are mean ± SEM; ∗P < 0.05, (Student's t-test). (C) IF with UCP1/red fluorescence secondary antibodies and perilipin1/green fluorescence secondary antibodies. Arrow: beige AT. (D) IF analysis with UCP1/red fluorescence secondary antibodies and co-stained with isolectin B4 (green) marking endothelium. Arrow: beige AT. (E) IF with phosphorylated hormone-sensitive lipase (pHSL)/red-fluorescent secondary antibodies and perilipin1/green-fluorescent secondary antibodies. Note browning (UCP1 expression) and lipolysis (pHSL expression, arrows) in BAT and tumor-proximal AT, which is reduced by D-BAT treatment but not by PBS. Nuclei are blue. Scale bars: 50 μm.

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  Figure 2. Endothelial TrkA regulates AT neuro-vascular alignment and thermogenesis. (A) SAT explants at day 4 of culture show normal angiogenic sprouting (arrows) for TrkA EC KO. (B) Whole mounts subjected to IF demonstrating blood vessel (endomucin+) misalignment with nerves (TH+) in BAT and SAT of KO mice. Graphs: data quantification; Error bars: SEM ∗P < 0.05, ∗∗P < 0.01 (Student's t-test); N = 5 view fields. (C) Paraffin sections stained with IB4 and subjected to UCP1 IF demonstrating lower UCP1 expression in BAT and lack of beige AT (arrows) in SAT of KO mice. Graphs: relative UCP1 expression quantification; ∗P < 0.01, N = 5 view fields. Error bars: SD ∗P < 0.05 (Student's t-test). In A-C, DNA is blue; Scale bar: 50 μm. (D) Cold intolerance of male KO mice vs WT littermates, measured based on core body temperature maintenance at 4 °C. N = 6. Error bars: SD ∗P < 0.05 (Student's t-test). (E) Reduced oxygen consumption of EC KO mice during day and night. N = 5. Error bars: SEM ∗P < 0.05 (Student's t-test) calculated based on the analysis of multiple time points (Supplementary Fig. 2D). (F) A model of TrkA function in AT endothelium. NGF secreted by brown adipocytes activates TrkA in both…