Endothelial PDGF-CC regulates angiogenesis-dependent thermogenesis in beige fat

Abstract

Cold- and β3-adrenoceptor agonist-induced sympathetic activation leads to angiogenesis and UCP1-dependent thermogenesis in mouse brown and white adipose tissues. Here we show that endothelial production of PDGF-CC during white adipose tissue (WAT) angiogenesis regulates WAT browning. We find that genetic deletion of endothelial VEGFR2, knockout of the Pdgf-c gene or pharmacological blockade of PDGFR-α impair the WAT-beige transition. We further show that PDGF-CC stimulation upregulates UCP1 expression and acquisition of a beige phenotype in differentiated mouse WAT-PDGFR-α(+) progenitor cells, as well as in human WAT-PDGFR-α(+) adipocytes, supporting the physiological relevance of our findings. Our data reveal a paracrine mechanism by which angiogenic endothelial cells modulate adipocyte metabolism, which may provide new targets for the treatment of obesity and related metabolic diseases.

Figure 1. β3-adrenoceptor-dependent activation of adipose angiogenesis and the beige phenotype in visceral adipose tissue.

(a) Microvessels (CD31⁺ red), adipocyte morphology (H&E; Perilipin⁺ green), and mitochondrial staining (Prohibitin⁺ red) of buffer-treated control (vehicle) and CL-treated gWAT in wild-type (wt) and Adrb3^−/− mice. White arrows and arrowheads point to microvessels and double-arrowed bars indicate adipocyte diameter. Yellow arrows point to prohibitin-positive signals. Cold (4 °C)- and thermoneutral temperature (30 °C)-exposed gWAT served as controls. n=5 mice for each group. (b) Quantification of microvessel density in various agent- or temperature-treated groups (n=10 random fields; n=5 mice for each group). (c) Quantification of average adipocyte size (>30 adipocytes per field; n=10 random fields; n=5 mice for each group). (d) Quantification of prohibitin-positive signals in various agents- or temperature-treated groups (n=10 random fields; n=5 mice for each group). (e) Vegf mRNA and protein expression in vehicle- and CL-treated gWAT samples. Total adipose tissue, MAF and SVF were used as starting materials (n=5 samples for each group). (f) UCP1 staining of gWAT in various agents- or temperature-treated wt and Adrb3^−/− mice. (n=5 samples for each group). Arrowheads point to UCP1-positive signals. Red=UCP1; green=perilipin; blue=DAPI. (g) Quantification of UCP1-positive staining signals (left, n=10 random fields) and mRNA (right, n=6–8 samples for each group). All scale bars, 50 μm. **P<0.01; ***P<0.001 by two-sided unpaired t-test. Data presented as mean±s.e.m. DAPI, 4',6-diamidino-2-phenylindole. n.s., not significant.

Figure 2. CL induces UCP1-independent adipose angiogenesis and upregulates VEGFR expression levels.

(a) Microvessels (CD31⁺ red), adipocyte morphology (H&E; perilipin⁺ green), and UCP1 expression (UCP1⁺ red) of vehicle- and CL-gWAT in wt and Ucp1^−/− mice. White arrows and arrowheads in upper three panels point to microvessels. Arrows in the lowest row of panels indicate UCP1-positive signals. Double-arrowed bars mark adipocyte diameter. n=5 mice for each wt and Ucp1^−/− group. (b) Quantification of microvessel density in vehicle- and CL-treated wt and Ucp1^−/− mice (n=10 random fields; n=5 mice for each group). (c) Quantification of the average adipocyte size (>30 adipocytes/field; n=10 random fields; n=5 mice for each group). (d) Vegf mRNA expression levels in vehicle- and CL-treated total gWAT (left) and MAF (right) samples in wt and Ucp1^−/− mice (n=10 samples for each group). (e) Quantification of UCP1-positive signals (n=10 random fields; n=5 mice for each group). (f) Vegfr1 and Vegfr2 mRNA expression levels in vehicle- and CL-treated total gWAT samples in wt mice (n=10 samples for each group). (g) FACS analysis of VEGFR1 and VEGFR2 expression levels in CD31⁺ and CD31⁻ ECs isolated from vehicle- and CL-treated gWAT in wt mice (n=5 samples for each group). All scale bars, 50 μm. **P<0.01; ***P<0.001 by two-sided unpaired t-test. Data presented as mean±s.e.m. n.s., not significant.

Figure 3. Effects of VEGF and VEGFR blockades on CL-induced adipose angiogenesis and beige phenotype.

(a) Microvessels (CD31⁺ red), adipocyte morphology (H&E; perilipin⁺ green) and UCP1 expression (UCP1⁺ red) of CL-treated gWAT in mice that were treated with vehicle, VEGF-, and VEGFR2-specific neutralizing antibodies (Anti-VEGF and Anti-R2) and sunitinib. The vehicle- treated group served as a control. White arrows and arrowheads in upper three panels point to microvessels. Arrows in the lowest row of panels indicate UCP1-positive signals. Double-arrowed bars mark adipocyte diameter. Green=perilipin; blue=DAPI. n=8 for each vehicle, VEGF and VEGFR blockades group. (b) Quantification of microvessel density in vehicle- and CL-treated wt mice that received treatment with various VEGF blockades (n=10 random fields; n=8 mice for each group). (c) Quantification of average adipocyte size (>30 adipocytes/field; n=10 random fields; n=8 mice for each group). (d) Quantification of UCP1-positive signals (n=10 random fields; n=8 mice for each group). (e) Norepinephrine-stimulated non-shivering thermogenesis in VEGF/VEGFR blockade- and vehicle-treated mice that received CL or buffer (n=5 mice for each group). All scale bars, 50 μm. *P<0.05; **P<0.01; ***P<0.001 by two-sided unpaired t-test. Data presented as mean±s.e.m. NE, norepinephrine.

Figure 4. Impaired CL-induced angiogenesis and beige transition in EC Vegfr2-specific eliminated mice.

(a) Microvessels (CD31⁺ red), adipocyte morphology (H&E; perilipin⁺ green), and UCP1 expression (UCP1⁺ red) of vehicle- and CL-treated gWAT in wt and Flk1f/f;Cdh5CreER^T2 mice. White arrows and arrowheads in upper three panels point to microvessels. Arrows in the lowest row of panels indicate UCP1-positive signals. Double-arrowed bars mark adipocyte diameter. n=5 for each wt and Flk1f/f;Cdh5CreER^T2 group. (b) Quantification of microvessel density in vehicle- and CL-treated wt and Flk1f/f;Cdh5CreER^T2 mice (n=10 random fields; n=5 mice for each group). (c) Quantification of average adipocyte size (>30 adipocytes/field; n=10 random fields; n=5 mice for each group). (d) Quantification of UCP1-positive signals (n=10 random fields; n=5 mice for each group). (e) qPCR quantification of mRNA expression levels Ucp1, Cidea, Cox8b, Pgc1α and Prdm16 in vehicle- and CL-treated total gWAT samples in wt and Flk1f/f;Cdh5CreER^T2 mice. (n=10 samples for each group). All scale bars, 50 μm. **P<0.01; ***P<0.001 by two-sided unpaired t-test. Data presented as mean±s.e.m.

Figure 5. Microarray and effect of AdVEGF delivery on adipose angiogenesis and CL-induced PDGF-C expression during beige transition.

(a) Histological images of microvessels (CD31⁺ red, white arrows and arrow heads), adipocyte morphology (H&E, double-arrowed bars, perilipin+green), and UCP1 (UCP1⁺ red, white arrows) in mice that were treated with AdGFP and AdVEGF for 10 days. The non-adenovirus-treated (NAT) group served as a control. n=5 mice for NAT group, n=10 mice for each AdGFP and AdVEGF group. (b) Quantification of Vegf mRNA expression levels in NAT- AdGFP- and AdVEGF-treated total gWAT. Quantification of microvessel density in NAT- AdGFP- and AdVEGF-treated (n=10 random fields; n=5 mice for NAT and n=10 for each AdGFP and AdVEGF group). (c) Quantification of UCP1-positive signals (n=10 random field; n=5 mice for NAT group and n=10 for each AdGFP and AdVEGF group) and quantification of Ucp1 mRNA expression levels by qPCR (n=10 samples for each group). (d) Quantification of average adipocyte size (>30 adipocytes/field; n=10 random fields; n=5 mice for NAT group and n=10 for each AdGFP and AdVEGF group). (e) Norepinephrine-stimulated non-shivering thermogenesis in AdGFP or AdVEGF-treated gWAT of mice (n=5 mice for each group). NE, norepinephrine. (f) Hierarchical clusters of top 20 growth factors and cytokines from genome-wide microarray analysis (n=3 samples for each group). qPCR analysis of Pdgf-c mRNA expression levels of vehicle- and CL-treated gWAT SVF (n=5 samples for each group). (g) Quantification of Pdgf-c mRNA expression levels in SVF by qPCR in AdGFP- and AdVEGF-treated total gWAT (n=10 samples). (h) QPCR analysis of Pdgf-a, -b, -c, and -d expression levels in total vehicle- and CL-treated CD31⁺ EC fraction from gWAT. (i) Pdgf-c mRNA expression levels in vehicle- and CL-treated total gWAT of wt and Flk1f/f;Tie2CreER^T2 mice. All scale bars, 50 μm. *P<0.05; **P<0.01 by two-sided unpaired t-test. Data presented as mean±s.e.m. n.s., not significant.

Figure 6. Impaired CL-induced beige transition in Pdgf-c^−/− mice and gain-of-function by delivery of AdPDGFC.

(a) Histological images of microvessels (CD31⁺ red, white arrows and arrow heads), adipocyte morphology (H&E, double arrowed bars, perilipin+green), and UCP1 (UCP1⁺ red, white arrows). n=5 mice for each wt and Pdgf-c^−/−group. (b) Quantification of microvessel density in vehicle- and CL-treated gWAT in wt and Pdgf-c^−/− mice. (n=10 random fields; n=5 mice for each group). (c) Quantification of average adipocyte size (>30 adipocytes/field; n=10 random fields; n=5 mice for each group). (d) Quantification of UCP1-positive signals (n=10 random fields; n=5 mice for each group). (e) qPCR quantification of browning markers' expression levels in vehicle- and CL-treated total gWAT samples in wt and Pdgf-c^−/− mice. (n=10 samples for each group). (f) Delivery of AdPDGFC to gWAT of CL-injected Pdgf-c^−/− mice. Arrows point to CD31⁺ microvessels and arrowheads point to UCP1-positive signals. Quantification of CD31⁺ and UCP1⁺ signals (n=10 randomized fields). n=8 mice for each wt and Pdgf-c^−/− group. (g) qPCR quantification of browning markers' expression levels in AdGFP- and AdPDGFC-injected gWAT of CL-treated Pdgf-c^−/−mice (n=12 samples for each group). (h) Delivery of AdPDGFC or AdGFP to gWAT of NOD-SCID mice. Arrows point to CD31⁺ microvessels and arrowheads point to UCP1-positive signals. (i) Quantification of CD31⁺ and UCP1⁺ signals (n=10 randomized fields; n=8 mice for each group). (j) Pdgf-c expression level in iWAT by delivery of AdPDGFC, body weight curve and adipose depot weights of HFD-induced obese mice receiving AdGFP and AdPDGFC treatment (n=6 animals for each group). (k) Insulin tolerance of AdGFP- and AdPDGFC-treated HFD-induced obese mice (n=10 animals for each group). The Mann–Whitney test. All scale bars, 50 μm. *P<0.05; **P<0.01; ***P<0.001 by two-sided unpaired t-test. Data presented as mean±s.e.m. n.s., not significant.

Figure 7. PDGF-CC promotes adipose progenitor cell differentiation toward a beige phenotype and PDGFR-α blockade CL-induced beige transition.

(a) Localization of PDGFR-α⁺ cells, the quantification, FACS analysis, and Pdgfra mRNA expression in vehicle- and CL-treated gWAT of wt mice (n=4 for staining and n=6 for FACS; n=10 for quantification; n=6 for qPCR). (b) qPCR analysis of browning markers' expression levels by PDGF-CC stimulation in differentiated gWAT-PDGFR-α⁺ cell populations. (c) Oil Red O staining and qPCR analysis (n=9 samples for each group) in human differentiated PDGFR-α⁻ and PDGFR-α⁺ cells. (d) Histological images of microvessels (CD31⁺ red, white arrows and arrow heads), adipocyte morphology (H&E, double arrowed bars; perilipin+green), and UCP1 (UCP1⁺ red, white arrows). (e) Quantification of microvessel density in vehicle- and CL-treated gWAT in wt mice that received PRα and PRβ blockade treatment (n=10 random fields; n=8 mice for each group). (f) Quantification of average gWAT adipocyte size (>30 adipocytes/field; n=10 random fields; n=8 mice for each group). (g) Quantification of UCP1-positive signals (n=10 random fields; n=8 mice for each group). (h) qPCR quantification of browning markers' expression levels in gWAT from wt mice that received various treatments. (i) Norepinephrine-stimulated non-shivering thermogenesis in PRα and PRβ blockade- and vehicle-treated mice that received CL or buffer (n=5 mice for each group). NE, norepinephrine. (j) Norepinephrine-stimulated non-shivering thermogenesis in PRα and PRβ blockade- and vehicle-treated mice that had been exposed to 4 or 30 °C (n=5 mice for each group). (k) Schematic diagram of paracrine regulatory mechanisms by which the VEGF-VEGFR2 and PDGF-CC-PDGFR-α signalling systems cohesively and reciprocally control adipose endothelial cell-adipocyte crosstalk, leading to the transition of beige cell differentiation from PDGFR-α⁺ cells in WAT. AR, adrenoceptor; BA, brown adipocyte; WA, white adipocyte. All scale bars, 50 μm. *P<0.05; **P<0.01; ***P<0.001 by two-sided unpaired t-test. Data presented as mean±s.e.m. n.s., not significant.