Paracrine regulation of angiogenesis and adipocyte differentiation during in vivo adipogenesis

Abstract

With an increasing incidence of obesity worldwide, rational strategies are needed to control adipogenesis. Growth of any tissue requires the formation of a functional and mature vasculature. To gain mechanistic insight into the link between active adipogenesis and angiogenesis, we developed a model to visualize noninvasively and in real time both angiogenesis and adipogenesis using intravital microscopy. Implanted murine preadipocytes induced vigorous angiogenesis and formed fat pads in a mouse dorsal skin-fold chamber. The newly formed vessels subsequently remodeled into a mature network consisting of arterioles, capillaries, and venules, whereas the preadipocytes differentiated into adipocytes as confirmed by increased aP2 expression. Inhibition of adipocyte differentiation by transfection of preadipocytes with a peroxisome proliferator-activated receptor gamma dominant-negative construct not only abrogated fat tissue formation but also reduced angiogenesis. Surprisingly, inhibition of angiogenesis by vascular endothelial growth factor receptor-2 (VEGFR2) blocking antibody not only reduced angiogenesis and tissue growth but also inhibited preadipocyte differentiation. We found that part of this inhibition stems from the paracrine interaction between endothelial cells and preadipocytes and that VEGF-VEGFR2 signaling in endothelial cells, but not preadipocytes, mediates this process. These findings reveal a reciprocal regulation of adipogenesis and angiogenesis, and suggest that blockade of VEGF signaling can inhibit in vivo adipose tissue formation. The full text of this article is available online at http://www.circresaha.org.

Figure 1

Angiogenesis and vessel remodeling during adipogenesis in the mouse dorsal skinfold chamber after 3T3-F442A cell implantation. A and B, Macroscopic images 9 days after implantation. C and D, Multiphoton laser-scanning microscopy images 28 days after preadipocyte implantation. Images were obtained by maximum intensity projection of 31 optical slices, each 5 µm thick: the top 150-µm de novo adipose tissue layer (C) and the bottom 150-µm host subcutaneous layer (D). E through H, High-power microscopic images of fluorescence contrast-enhanced blood vessels at 7 days (E), 14 days (F), 21 days (G), and 28 days (H) after implantation. Bars indicate 5 mm (A), 0.5 mm (B), 200 µm (C and D), and 100 µm (E through H), respectively.

Figure 2

Quantitative analysis of blood vessels during adipogenesis (n=7 mice). A, Number of vessel segments in the high-power view field. B, Vascular length density. C, Vessel diameter. D, Calculated blood vessel volume. E, Vessel diameter histogram. Each vessel segment was categorized by diameter and shown as a cumulative frequency distribution. Segment diameters were distributed over a wide range at day 7. Distribution shifted leftward and the range became narrow as a result of vessel remodeling with continued adipogenesis. At day 28, most segments (92%) were 3 to 9 µm in diameter.

Figure 3

Differentiation of 3T3-F442A preadipocytes and de novo adipose tissue formation: the effect of PPARγ-DN. A, Bright-field microscopic image of differentiated adipocytes in vitro. B, Macroscopic images of Oil Red O staining. C, Fluorescence image of differentiated adipocytes in vivo. To identify implanted cells in vivo, we used GFP-labeled preadipocytes. We found fat accumulation in GFP-positive cells during conversion of fibroblast-shaped preadipocytes to round-shaped adipocytes. This was visible as granular fluorescence due to fat droplets in the cytosol and was observed by multiphoton microscope (with rhodamine-dextran vessel enhancement). D, Active adipogenesis on a subcutaneous cell implant, confirmed by increasing aP2 levels (values shown after quantitative densitometry of a Northern blot analysis). E and F, Fluorescence microscopy in tissue sections from (E) mock- and (F) PPARγ-DN–transfected preadipocyte-generated tissues. G and H, Histological analyses of tissue expansion and cell size. G, An increased number of cells and tissue area is seen in mock preadipocyte tissue compared with PPARγ-DN tissue. H, Number of cells per tissue area is decreased in mock preadipocyte–generated tissue, documenting the increase in size of the cells in these tissues. *P<0.01 as compared with corresponding control by two-tailed t test; **P<0.05 using the Wilcoxon rank-sum test. Bars=50 µm (A), 50 µm (C), and 0.5 mm (E and F), respectively.

Figure 4

Effect of PPARγ-DN on angiogenesis in the adipose tissue generated from preadipocytes. A and B, Fluorescence images of blood vessels 21 days after mock- (A) and PPARγ-DN–transfected (B) preadipocyte implantation. Bar=100 µm. C through F, Quantitative analyses of tissue neovascularization: C, number of vessel segments; D, vascular length density; E, vessel diameter; F, vessel volume. Filled circles, control (n=6); open squares, PPARγ-dominant-negative–transfected preadipocytes (n=5). There was no difference between the two different sets of control cells (mock-transfected preadipocytes, n=3 mice, and EF1α-GFP 3T3-F442A cells, n=3). These two groups are combined for presentation and statistical analysis. *P<0.01 as compared with corresponding control by two-tailed t test.

Figure 5

Effect of VEGFR2 blockade on angiogenesis and adipogenesis. A and B, Visualization of rhodamine-dextran contrast-enhanced blood vessels 21 days after preadipocyte implantation with control IgG (A) and DC101 (B) treatments. C through F, Quantitative analyses of tissue neovascularization: C, number of vessel segments; D, vascular length density; E, vessel diameter; F, vessel volume. Filled circles represent IgG treatment (n=6 mice); open squares, DC101 treatment (n=6 mice). *P<0.01 as compared with IgG by two-tailed t test. G, In vivo gene expression of aP2 in preadipocyte-generated tissue with densitometry normalized to control condition (PBS).

Figure 6

Role of VEGFR2 signaling in preadipocyte differentiation and proliferation. A, Confluent 3T3-F442A cells were maintained for 11 days with either 10% calf serum (CS, maintenance media) with or without VEGF (50 ng/mL) or 10% FBS (differentiation media) and either DC101 or rat IgG (1 µg/mL). At day 11, cells were stained with Oil Red O. Mouse recombinant VEGF did not induce differentiation in preadipocytes cultured in 10% CS (maintenance media) and did not increase the differentiation rate in cells treated with 10% FBS (differentiation media). Addition of DC101 or IgG to the culture media had no effect on in vitro adipogenesis. Bar=50 µm. B, Mouse recombinant VEGF (50 ng/mL) and PBS, DC101, or rat IgG was added (concentration of the antibodies, 1 µg/mL), and the MTT assay was performed at day 4. The optical density values were normalized to that of the PBS-treated cells and were used as a measure of viability. C, MTT assay for preadipocytes after 4 days of culture with endothelial conditioned media [FBS indicates nonconditioned culture media with 10% FBS; SN, supernatant, endothelial conditioned media cultured with 10% FBS but no other additives; SN/VEGF/IgG, endothelial conditioned media cultured with 10% FBS, VEGF (50 ng/mL) and nonspecific IgG (5 µg/mL); SN/VEGF/DC101, endothelial conditioned media cultured with 10% FBS, VEGF (50 ng/mL) and DC101 (5 µg/mL)]. Data were normalized to the control condition (FBS). *P<0.01 as compared with SN/VEGF/DC101 by two-tailed t test. Error bars represent standard error. D, In vitro gene expression of aP2 in preadipocytes after 11 days of culture with endothelial conditioned media (VEGF/IgG, endothelial conditioned media cultured with 10% FBS, VEGF, and nonspecific IgG; SN, endothelial conditioned media with 10% FBS and no other additives; VEGF/DC101, endothelial conditioned media cultured with 10% FBS, VEGF, and DC101). Fold increase in aP2 expression by differentiating preadipocytes was calculated by densitometry and normalized to the control condition (SN).