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Intravitreally Injected Anti-VEGF Antibody Reduces Brown Fat in Neonatal Mice.

Jo DH, Park SW, Cho CS, Powner MB, Kim JH, Fruttiger M, Kim JH - PLoS ONE (2015)

Bottom Line: Furthermore, in interscapular brown adipose tissue, we found lipid droplet accumulation, decreased VEGF levels, loss of vascular network, and decreased expression of mitochondria-related genes, Ppargc1a and Ucp1, all of which are characteristics of "whitening" of brown fat.With increasing age and body weight, brown fat restored its morphology and vascularity.We suggest that more attention should be focused on the metabolic and developmental significance of brown adipose tissue in bevacizumab treated retinopathy of prematurity infants.

View Article: PubMed Central - PubMed

Affiliation: Fight against Angiogenesis-Related Blindness (FARB) Laboratory, Clinical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea; Department of Biomedical Sciences, College of Medicine, Seoul National University, Seoul, Republic of Korea.

ABSTRACT
Anti-vascular endothelial growth factor (VEGF) agents are the mainstay treatment for various angiogenesis-related retinal diseases. Currently, bevacizumab, a recombinant humanized anti-VEGF antibody, is trailed in retinopathy of prematurity, a vasoproliferative retinal disorder in premature infants. However, the risks of systemic complications after intravitreal injection of anti-VEGF antibody in infants are not well understood. In this study, we show that intravitreally injected anti-VEGF antibody is transported into the systemic circulation into the periphery where it reduces brown fat in neonatal C57BL/6 mice. A considerable amount of anti-VEGF antibody was detected in serum after intravitreal injection. Furthermore, in interscapular brown adipose tissue, we found lipid droplet accumulation, decreased VEGF levels, loss of vascular network, and decreased expression of mitochondria-related genes, Ppargc1a and Ucp1, all of which are characteristics of "whitening" of brown fat. With increasing age and body weight, brown fat restored its morphology and vascularity. Our results show that there is a transient, but significant impact of intravitreally administered anti-VEGF antibody on brown adipose tissue in neonatal mice. We suggest that more attention should be focused on the metabolic and developmental significance of brown adipose tissue in bevacizumab treated retinopathy of prematurity infants.

No MeSH data available.


Related in: MedlinePlus

Effects of intravitreally injected anti-VEGF antibody on BAT of neonatal mice.(A) Concentrations of VEGF in interscapular BAT at P21 and P28. The level of VEGF was normalized to total amounts of proteins in BAT (n = 3–6). (B) Representative images of H&E staining of interscapular BAT after intravitreal injection of PBS or anti-VEGF antibody show enlarged lipid droplets. Scale bar, 20 μm. (C) Quantitative analyses of vascularity of interscapular BAT based on isolectin B4 staining (n = 3–6). The effects of anti-VEGF antibody were quantified and normalized to the control (intravitreal PBS injection). (D) Relative expression of Ucp1 and Ppargc1a in interscapular BAT (n = 3–6). Data are presented as mean ± SEM in graphs. Anti-VEGF, anti-VEGF antibody. *, P < 0.05; **, P < 0.01; ***, P <0.001 (two-tailed, unpaired T-test).
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pone.0134308.g002: Effects of intravitreally injected anti-VEGF antibody on BAT of neonatal mice.(A) Concentrations of VEGF in interscapular BAT at P21 and P28. The level of VEGF was normalized to total amounts of proteins in BAT (n = 3–6). (B) Representative images of H&E staining of interscapular BAT after intravitreal injection of PBS or anti-VEGF antibody show enlarged lipid droplets. Scale bar, 20 μm. (C) Quantitative analyses of vascularity of interscapular BAT based on isolectin B4 staining (n = 3–6). The effects of anti-VEGF antibody were quantified and normalized to the control (intravitreal PBS injection). (D) Relative expression of Ucp1 and Ppargc1a in interscapular BAT (n = 3–6). Data are presented as mean ± SEM in graphs. Anti-VEGF, anti-VEGF antibody. *, P < 0.05; **, P < 0.01; ***, P <0.001 (two-tailed, unpaired T-test).

Mentions: Next, we examined VEGF levels in interscapular BAT at P21 and P28, 1 and 2 weeks after intravitreal injection, respectively. It is notable that the size of interscapular BAT were not significantly changed (data not shown). In contrast, at both time points, intravitreally injected anti-VEGF antibody significantly reduces the level of VEGF in BAT (Fig 2A; P-value = 0.0317 and 0.0086, respectively). We speculated that the reduction in the level of VEGF might be due to direct scavenging of VEGF by anti-VEGF antibody. Targeted genetic deletion of VEGF in adipose tissue has been previously shown to induce indicators of BAT “whitening”, such as lipid droplet accumulation and mitochondrial dysfunction [15]. In keeping with this, in our study, H&E staining demonstrated that anti-VEGF antibody dramatically increased the number of large lipid droplets (> 50 μm2) in BAT (Fig 2B and S2A Fig). BAT is metabolically active and displays higher vascular density [14]. Moreover, VEGF-A is a key factor in governing angiogenesis in adipose tissue [27]. We therefore evaluated the effects of anti-VEGF antibody on vascular network in BAT and found a clear reduction in vessel density, as demonstrated by isolectin B4 staining (Fig 2C and S2B Fig).


Intravitreally Injected Anti-VEGF Antibody Reduces Brown Fat in Neonatal Mice.

Jo DH, Park SW, Cho CS, Powner MB, Kim JH, Fruttiger M, Kim JH - PLoS ONE (2015)

Effects of intravitreally injected anti-VEGF antibody on BAT of neonatal mice.(A) Concentrations of VEGF in interscapular BAT at P21 and P28. The level of VEGF was normalized to total amounts of proteins in BAT (n = 3–6). (B) Representative images of H&E staining of interscapular BAT after intravitreal injection of PBS or anti-VEGF antibody show enlarged lipid droplets. Scale bar, 20 μm. (C) Quantitative analyses of vascularity of interscapular BAT based on isolectin B4 staining (n = 3–6). The effects of anti-VEGF antibody were quantified and normalized to the control (intravitreal PBS injection). (D) Relative expression of Ucp1 and Ppargc1a in interscapular BAT (n = 3–6). Data are presented as mean ± SEM in graphs. Anti-VEGF, anti-VEGF antibody. *, P < 0.05; **, P < 0.01; ***, P <0.001 (two-tailed, unpaired T-test).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4520452&req=5

pone.0134308.g002: Effects of intravitreally injected anti-VEGF antibody on BAT of neonatal mice.(A) Concentrations of VEGF in interscapular BAT at P21 and P28. The level of VEGF was normalized to total amounts of proteins in BAT (n = 3–6). (B) Representative images of H&E staining of interscapular BAT after intravitreal injection of PBS or anti-VEGF antibody show enlarged lipid droplets. Scale bar, 20 μm. (C) Quantitative analyses of vascularity of interscapular BAT based on isolectin B4 staining (n = 3–6). The effects of anti-VEGF antibody were quantified and normalized to the control (intravitreal PBS injection). (D) Relative expression of Ucp1 and Ppargc1a in interscapular BAT (n = 3–6). Data are presented as mean ± SEM in graphs. Anti-VEGF, anti-VEGF antibody. *, P < 0.05; **, P < 0.01; ***, P <0.001 (two-tailed, unpaired T-test).
Mentions: Next, we examined VEGF levels in interscapular BAT at P21 and P28, 1 and 2 weeks after intravitreal injection, respectively. It is notable that the size of interscapular BAT were not significantly changed (data not shown). In contrast, at both time points, intravitreally injected anti-VEGF antibody significantly reduces the level of VEGF in BAT (Fig 2A; P-value = 0.0317 and 0.0086, respectively). We speculated that the reduction in the level of VEGF might be due to direct scavenging of VEGF by anti-VEGF antibody. Targeted genetic deletion of VEGF in adipose tissue has been previously shown to induce indicators of BAT “whitening”, such as lipid droplet accumulation and mitochondrial dysfunction [15]. In keeping with this, in our study, H&E staining demonstrated that anti-VEGF antibody dramatically increased the number of large lipid droplets (> 50 μm2) in BAT (Fig 2B and S2A Fig). BAT is metabolically active and displays higher vascular density [14]. Moreover, VEGF-A is a key factor in governing angiogenesis in adipose tissue [27]. We therefore evaluated the effects of anti-VEGF antibody on vascular network in BAT and found a clear reduction in vessel density, as demonstrated by isolectin B4 staining (Fig 2C and S2B Fig).

Bottom Line: Furthermore, in interscapular brown adipose tissue, we found lipid droplet accumulation, decreased VEGF levels, loss of vascular network, and decreased expression of mitochondria-related genes, Ppargc1a and Ucp1, all of which are characteristics of "whitening" of brown fat.With increasing age and body weight, brown fat restored its morphology and vascularity.We suggest that more attention should be focused on the metabolic and developmental significance of brown adipose tissue in bevacizumab treated retinopathy of prematurity infants.

View Article: PubMed Central - PubMed

Affiliation: Fight against Angiogenesis-Related Blindness (FARB) Laboratory, Clinical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea; Department of Biomedical Sciences, College of Medicine, Seoul National University, Seoul, Republic of Korea.

ABSTRACT
Anti-vascular endothelial growth factor (VEGF) agents are the mainstay treatment for various angiogenesis-related retinal diseases. Currently, bevacizumab, a recombinant humanized anti-VEGF antibody, is trailed in retinopathy of prematurity, a vasoproliferative retinal disorder in premature infants. However, the risks of systemic complications after intravitreal injection of anti-VEGF antibody in infants are not well understood. In this study, we show that intravitreally injected anti-VEGF antibody is transported into the systemic circulation into the periphery where it reduces brown fat in neonatal C57BL/6 mice. A considerable amount of anti-VEGF antibody was detected in serum after intravitreal injection. Furthermore, in interscapular brown adipose tissue, we found lipid droplet accumulation, decreased VEGF levels, loss of vascular network, and decreased expression of mitochondria-related genes, Ppargc1a and Ucp1, all of which are characteristics of "whitening" of brown fat. With increasing age and body weight, brown fat restored its morphology and vascularity. Our results show that there is a transient, but significant impact of intravitreally administered anti-VEGF antibody on brown adipose tissue in neonatal mice. We suggest that more attention should be focused on the metabolic and developmental significance of brown adipose tissue in bevacizumab treated retinopathy of prematurity infants.

No MeSH data available.


Related in: MedlinePlus