Deficiency of vascular endothelial growth factor-D does not affect murine adipose tissue development

Biochemical and Biophysical Research Communications 378 (2009) 255–258

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Deficiency of vascular endothelial growth factor-D does not affect murine adipose tissue development H.R. Lijnen a,*, L. Frederix a, B. Van Hoef a, M. Dewerchin b a b

Center for Molecular and Vascular Biology, KU Leuven, Campus Gasthuisberg, O & N 1, Herestraat 49, Box 911, 3000 Leuven, Belgium Vesalius Research Center, VIB, Campus Gasthuisberg, O&N 1, Herestraat 49, Box 914, 3000 Leuven, Belgium

a r t i c l e

i n f o

Article history: Received 17 October 2008 Available online 18 November 2008

Keywords: Obesity Adipose tissue Lymphangiogenesis VEGF-D

a b s t r a c t Vascular endothelial growth factor (VEGF)-D deficiency had no significant effect on total body weight or on subcutaneous (SC) or gonadal (GON) adipose tissue mass of mice kept on a standard fat (SFD) or a high fat diet (HFD) for 15 weeks. The composition of SC and GON adipose tissues of VEGF-D deficient mice in terms of size and density of adipocytes or blood vessels was also comparable to that of wild-type control mice. Staining of lymphatic vessels in adipose tissue sections did not reveal marked differences between both genotypes. The absence of an effect of VEGF-D deficiency could not be explained by compensatory increases of VEGF-C expression in adipose tissues of the deficient mice. Thus, our data do not support an important role of VEGF-D in (lymph) angiogenesis or in adipose tissue development. Ó 2008 Elsevier Inc. All rights reserved.

It is well established that development of obesity is associated with extracellular matrix proteolysis, adipogenesis and angiogenesis [1]. Furthermore, a link was established between lymph fluid and fat deposition, and it was shown that lymphatic vascular defects may cause adult-onset obesity [2,3]. Vascular endothelial growth factors (VEGF) are considered to be the main angiogenic components in adipose tissue [4]. VEGF-C and VEGF-D are VEGF homologs which bind VEGF-R3 on lymphatic endothelial cells [5– 7]. VEGF-C deficiency impairs lymphatic endothelial cell sprouting in the embryo [8], whereas VEGF-D does not appear to be relevant for development in mice ([9] and unpublished results). Studies on cancer and cancer metastasis have implicated both VEGF-C and VEGF-D in pathological lymphangiogenesis [10]. VEGF-D is indeed expressed in aggressive tumors [11,12] and overexpression of VEGF-D increased lymphangiogenesis in cancer [13,14]. In this study we have evaluated the potential contribution of VEGF-D dependent lymphangiogenesis in development of murine adipose tissue. Materials and methods Animals and models. VEGF-D null mice were created by deletion of exon 2–4 of the gene; the generation and characterization of the mice will be described elsewhere. Because of the X-chromosomal location of the VEGF-D gene, the VEGF-D deficient colony was established using wild-type (VEGF-D0/+) and hemizygous null

* Corresponding author. Fax: +32 16 345990. E-mail address: [email protected] (H.R. Lijnen). 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.11.032

(VEGF-D0/ ) males mated to heterozygous (VEGF-D+/ ) females to obtain the different genotypes, as monitored by PCR genotyping. Five weeks old male mice (VEGF-D0/+ or VEGF-D0/ ) were kept on a high fat diet (HFD) for 15 weeks (TD88137, Harlan Teklad Zeist, The Netherlands, containing 42% kcal as fat with a caloric value of 20.1 kJ per g) or on a standard fat diet (SFD) (KM-04-k12, Muracon, Carfil, Oud-Turnhout, Belgium, containing 13% kcal as fat with a caloric value of 10.9 kJ per g). Mice were kept in microisolation cages on a 12 h day/night cycle and were fed at libitum. At the end of the experiment, the mice were killed by i.p. injection of 60 mg/kg sodium pentobarbital (Abbott Laboratories, North Chicago, IL). Blood was collected from the retroorbital sinus on trisodium citrate (final concentration 0.01 mol/L) and plasma was stored at 20 °C. Epididymal (gonadal, GON) and inguinal (subcutaneous, SC) fat pads were removed and weighed; portions were immediately frozen at 80 °C for extraction and other portions were used to prepare 10 lm paraffine sections for histology [15]. Food intake was measured weekly for 4 day periods throughout the experimental period, and expressed as g per mouse and per day. All animal experiments were approved by the local Ethical Committee (KU Leuven, P03112) and performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals (1996) and the guiding principles of the International Society on Thrombosis and Haemostasis [16]. Assays. Adipose tissue sections were stained with haematoxylin/ eosin using a standard protocol or with the biotinylated Bandeiraea (Griffonia) Simplicifolia BSI lectin (Sigma–Aldrich, Bornem, Belgium) followed by signal amplification with the Tyramide Signal Amplification Cyanine System (Perkin Elmer, Boston, MA). The

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and shown as DCt values. This method does not allow quantitative comparison of VEGF-C with VEGF-D expression levels. Statistical analysis. Data are expressed as means ± SEM. Statistical significance for differences between groups, analyzed by nonparametric Mann–Whitney U-testing, was set at p < 0.05. Results The body weight gain was comparable for VEGF-D0/+ and VEGFmice, both on SFD and HFD. This resulted in comparable total D body weights at the end of the study for both genotypes on SFD or on HFD (Fig 1, Table 1). VEGF-D deficiency had no effect on food intake when the mice were kept on HFD (3.12 ± 0.11 versus 3.51 ± 0.11 g/mouse/day for VEGF-D0/+; p = 0.12), whereas on SFD the food intake of the VEGF-D0/ mice was higher (4.43 ± 0.06 versus 3.97 ± 0.04 g/mouse/day for VEGF-D0/+; p = 0.003). The feeding efficiency (weight gain normalized to caloric intake) thus was not different on the HFD (average 3.6 mg/kJ for both genotypes) but was higher for VEGF-D0/ mice on SFD (1.1 versus 1.4 mg/ kJ).VEGF-D deficiency did not significantly affect development of SC or GON adipose tissue on SFD or HFD. The weight of other organs was also comparable, indicating that VEGF-D deficiency did not affect general development of the mice. Analysis of adipocyte and blood vessel size and density in SC and GON adipose tissues did not reveal a significant effect of VEGF-D deficiency for mice on either diet (Table 2). Overall, the increase in body weight, SC and GON fat mass, adipocyte and blood vessel size observed in VEGF-D0/ mice when fed a HFD as com0/

Fig. 1. Evolution of body weight of VEGF-D0/+ (open symbols) or VEGF-D0/ (closed symbols) mice kept on SFD (squares) or HFD (circles). Data are means ± SEM of 9 or 10 experiments.

number of adipocytes and their mean size were determined by computer-assisted image analysis, using for each animal three to five areas in four different sections; the data were first averaged per section and then per animal. The lectin staining allows to visualize blood vessels [17]; average blood vessel size was calculated by dividing the total stained area by the number of vessels, and blood vessel density was expressed as the number of vessels per measured surface area ([9–12] sections were analyzed per animal and then averaged). Lymphatic vessels were stained using the LYVE-1 antibody (Upstate), and analyzed using the KS300 morphometric software (Zeiss). Double immunostaining with LYVE-1 and with the Bandeiraea lectin was performed to confirm specificity of the staining. Blood glucose concentrations were determined using Glucocard strips (Menarini Diagnostics, Florence, Italy); triglyceride, total, HDL and LDL cholesterol levels as well as liver enzymes (AST, ALT) were evaluated using routine clinical assays. PAI-1 antigen levels were measured with a specific home-made ELISA [18]. Insulin (Mercodia, Uppsala, Sweden) and leptin (R&D Systems Europe, Lille, France) were measured with commercially available ELISAs. Expression of VEGF-C in extracts of SC or GON adipose tissues was monitored by real-time PCR, as described [19]. VEGF-D expression was monitored with the Geneassay Mm003-438965_ml, using GADPH (Geneassay Mm99999915_gl) as internal control (Applied Biosystems). Ct values are normalized to the housekeeping gene

Table 2 Effect of VEGF-D deficiency on adipocyte and blood vessel size and density in adipose tissue of mice kept on SFD or HFD. SFD

HFD

VEGF-D0/+

VEGF-D0/

VEGF-D0/+

VEGF-D0/

2295 ± 150 3695 ± 350

4250 ± 230 6875 ± 315

3580 ± 245 6110 ± 260

490 ± 34 295 ± 28

240 ± 13 150 ± 6.8

295 ± 23 170 ± 7.3

52 ± 3.6 60 ± 2.9

63 ± 4.5 80 ± 5.8

57 ± 3.1 83 ± 8.4

510 ± 41 290 ± 26

365 ± 18 270 ± 23

400 ± 23 310 ± 23

2

Adipocyte size (lm ) SC fat 2190 ± 270 GON fat 3960 ± 320 Adipocyte density (106/lm2) SC fat 530 ± 51 GON fat 270 ± 20 Blood vessel size (lm2) SC fat 46 ± 2.3 GON fat 57 ± 3.4 Blood vessel density (106/lm2) SC fat 480 ± 33 GON fat 265 ± 12 Data are means± SEM.

Table 1 Effect of VEGF-D deficiency on adipose tissue and organ weights of mice kept on SFD or HFD. SFD

Body weight start (g) Body weight enda (g) SC fat (mg) GON fat (mg) Liver (mg) Kidney (mg) Spleen (mg) Pancreas (mg) Lung (mg) Heart (mg) Data are means ± SEM of n experiments. a Body weight after overnight fasting.

HFD

VEGF-D0/+ (n = 10)

VEGF-D0/ (n = 10)

VEGF-D0/+ (n = 10)

VEGF-D0/ (n = 9)

20 ± 0.40 30 ± 0.60 550 ± 60 820 ± 71 1240 ± 39 190 ± 6.5 76 ± 2.9 250 ± 4.4 140 ± 2.4 150 ± 4.2

20 ± 0.58 28 ± 0.66 410 ± 42 760 ± 84 1180 ± 56 190 ± 5.0 71 ± 3.0 230 ± 22 150 ± 4.0 150 ± 5.0

20 ± 0.46 44 ± 1.1 1575 ± 89 2330 ± 150 4500 ± 280 210 ± 8.2 120 ± 9.2 310 ± 16 160 ± 5.5 170 ± 4.3

20 ± 0.27 42 ± 1.4 1470 ± 125 2290 ± 80 3660 ± 380 210 ± 5.0 110 ± 6.0 280 ± 10 160 ± 2.0 170 ± 5.0

H.R. Lijnen et al. / Biochemical and Biophysical Research Communications 378 (2009) 255–258 Table 3 Expression of VEGF-C and VEGF-D in adipose tissues of mice kept on SFD or HFD. VEGF-C SFD

VEGF-D HFD

SFD

HFD

VEGF-D0/+ mice SC fat 11.8 ± 0.22 GON fat 10.5 ± 0.24

12.2 ± 0.19 11.5 ± 0.12*

5.54 ± 0.24 4.90 ± 0.20

5.05 ± 0.12 4.47 ± 0.32

VEGF-D0/ mice SC fat 13.0 ± 0.29+ GON fat 11.3 ± 0.29

12.1 ± 0.14* 11.3 ± 0.31

— —

— —

Data represent DCt values, normalized to GAPDH expression, and are expressed as means ± SEM. * p < 0.05 versus SFD. + p < 0.01 versus VEGF-D0/+.

pared to a SFD, was very comparable to VEGF-D0/+ mice. Furthermore, staining of lymph vessels in adipose tissue sections with the LYVE-1 antibody did not reveal apparent differences between both genotypes (not shown). VEGF-D0/+ mice kept on HFD, as compared to SFD, showed only a slight increase in VEGF-D expression in SC (1.4-fold) or GON (1.3fold) adipose tissues (p = NS) (Table 3). VEGF-C expression upon HFD feeding of VEGF-D0/+ mice was reduced, as compared to SFD in SC (1.3-fold; p = NS) or GON (2.0)-fold; p = 0.016) adipose tissues. For VEGF-D0/ mice, VEGF-C mRNA levels on HFD were higher for SC (1.8-fold; p = 0.016) but not for GON adipose tissues. Comparing VEGF-C expression levels between genotypes thus revealed downregulation in VEGF-D0/ mice on SFD (2.3-fold for SC, p = 0.008, and 1.7-fold, p = 0.09 for GON fat). On HFD, VEGF-C mRNA levels were comparable for SC and GON adipose tissues for both genotypes (Table 3). Analysis of plasma metabolic parameters did not show a significant effect of VEGF-D deficiency with SFD or HFD feeding on glucose, cholesterol or triglycerides, nor on alkaline phosphatase, AST or ALT levels (Table 4). Insulin and leptin levels in both genotypes increased on HFD as compared to SFD, but remained significantly lower in the HFD fed VEGF-D0/ mice. Plasma PAI-1 levels also strongly increased upon HFD feeding, and remained somewhat lower in the VEGF-D0/ mice. The impact of the HFD on the VEGF-D0/ mice thus was very comparable to the VEGF-D0/+ controls. Discussion VEGF-D is mitogenic for endothelial cells and has angiogenic as well as lymphangiogenic potential in vivo [20]. VEGF-D is closely related to VEGF-C by the presence of NH2 and COOH-terminal

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extensions that are lacking in other VEGF family members [21,22]. It stimulates growth of vascular and lymphatic endothelial cells by signaling through the tyrosine kinase receptors VEGFR-2 and VEGFR-3 [23]. In experimental tumors VEGF-D plays a role in stimulating lymphangiogenesis and lymphatic metastasis [24]. The observation that mice lacking Prox1, a gene required for the formation of lymphatic endothelial cells, display adult-onset obesity indicates a link between lymph fluid and fat deposition [2,3]. Leakage of lymph from abnormally formed lymph vessels in these mice stimulated cells to store fat [2]. Adipogenesis and fat deposition are known to occur efficiently around lymph nodes, where the adipocytes provide energy for local lymphoid metabolic needs [23]. Presumably the adipogenic effects of lymph are directly related to the development of obesity in the Prox1 deficient mice [2,3]. These finding suggest that lymphangiogenesis, in addition to angiogenesis, may play an important role in development of adipose tissue. Expression of the VEGF family members in adipocytes and adipose tissue of obese mice has been documented [19]. Therefore, we hypothesized that VEGF-D, because of its potential role in lymphangiogenesis, may play a role in development of adipose tissue. The availability of mice deficient in VEGF-D has allowed to test this hypothesis experimentally, using a well-established model of nutritionally induced obesity [15]. We found, however, that VEGF-D0/ mice when kept on HFD developed adipose tissue to a comparable extent as their wild-type littermates. The composition of SC and GON adipose tissues of both genotypes in terms of adipocyte size and density was very similar. Furthermore, no significant differences were observed in adipose tissue associated angiogenesis between VEGF-D0/ and VEGF-D0/+ mice kept on SFD or HFD, as revealed by comparable size and density of blood vessels. This is in agreement with the lack of binding of murine VEGF-D, unlike human VEGF-D, to the blood endothelial receptor VEGFR-2 [25]. LYVE-1 staining also did not establish differences in lymphangiogenesis. These negative findings could be explained by assuming that VEGF-D mediated lymphangiogenesis does not play an important role in adipose tissue development. It cannot be excluded that VEGF-C induced lymphangiogenesis suffices or compensates for the absence of VEGF-D. Expression of VEGF-C was actually found to be lower in adipose tissues of lean (SFD) VEGF-D0/ as compared to VEGF-D0/+ mice, and comparable for obese (HFD) mice of both genotypes, indicating that no compensatory increase in VEGF-C occurred in VEGF-D0/ mice. The HFD induced a marked increase in plasma levels of insulin and leptin, which remained significantly lower in VEGF-D0/ mice as compared to VEGF-D0/+ mice. Leptin was reported to have a synergistic effect on stimulation of angiogenesis by VEGF or FGF-2

Table 4 Effect of VEGF-D deficiency on metabolic parameters and liver enzymes of mice kept on SFD or HFD. SFD

PAI-1 (ng/mL) Glucose (mg/dL) Insulin (ng/mL) Leptin (ng/mL) Total cholesterol (mg/dL) HDL cholesterol (mg/dL) LDL cholesterol (mg/dL) Triglycerides (mg/dL) Alkaline phosphatases (U/L) AST (U/L) ALT (U/L)

VEGF-D0/

VEGF-D0/+

VEGF-D0/

3.1 ± 0.29 110 ± 8.5 0.91 ± 0.12 3.4 ± 1.1 71 ± 1.3 65 ± 1.7 N.D. 30 ± 1.5 150 ± 6.2 77 ± 5.6 44 ± 3.9

3.1 ± 0.31 120 ± 21 0.75 ± 0.13 3.3 ± 0.72 63 ± 2.4 57 ± 2.5 N.D. 44 ± 3.3 140 ± 4.4 70 ± 6.5 37 ± 3.5

20 ± 2.2 240 ± 33 7.1 ± 1.0 38 ± 3.0 250 ± 17 190 ± 10 48 ± 7.7 32 ± 3.1 310 ± 34 270 ± 27 400 ± 49

14 ± 3.2 190 ± 11 4.8 ± 0.62* 26 ± 2.2** 210 ± 18 170 ± 9.4 37 ± 8.6 27 ± 2.6 270 ± 36 220 ± 40 250 ± 68

Data are means ± SEM of 9 or 10 determinations; N.D., not detectable. p < 0.05 versus VEGF-D0/+. ** p < 0.01 versus VEGF-D0/+. *

HFD

VEGF-D0/+

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[26]. We did, however, not observe a difference in blood vessel size or density in adipose tissues of either genotype on HFD, likely due to the inability of murine VEGF-D to bind VEGFR-2 [25]. The elevated insulin levels on HFD may also contribute to the enhanced PAI-1 levels, as insulin is known to promote PAI-1 production by adipose tissue [27]. In summary, a nutritionally induced obesity model in VEGF-D deficient mice has not revealed an important role of VEGF-D in (lymph) angiogenesis or in adipose tissue development. Our data, however, do not allow to conclude that lymphangiogenesis would not play a role in adipose tissue development. Acknowledgments This study was supported financially by the ‘‘Fonds voor Wetenschappelijk Onderzoek-Vlaanderen” (G.0281.04) and by the Interuniversity Attraction Poles (IUAP, P6/30). The Center for Molecular and Vascular Biology is supported by the ‘‘Excellentiefinanciering KULeuven” (Project EF/05/013). References [1] D.L. Crandall, G.J. Hausman, J.G. Kral, A review of the microcirculation of adipose tissue: anatomic, metabolic, and angiogenic perspectives, Microcirculation 4 (1997) 211–232. [2] N.L. Harvey, R.S. Srinivasan, M.E. Dillard, N.C. Johnson, M.H. Witte, K. Boyd, M.W. Sleeman, G. Oliver, Lymphatic vascular defects promoted by Prox1 haploinsufficiency cause adult-onset obesity, Nat. Genet. 37 (2005) 1072–1081. [3] M. Schneider, E.M. Conway, P. Carmeliet, Lymph makes you fat, Nat. Genet. 37 (2005) 1023–1024. [4] G.J. Hausman, R.L. Richardson, Adipose tissue angiogenesis, J. Anim. Sci. 82 (2004) 925–934. [5] K. Alitalo, T. Tammela, T.V. Petrova, Lymphangiogenesis in development and human disease, Nature 438 (2005) 946–953. [6] T. Tammela, B. Enholm, K. Alitalo, K. Paavonen, The biology of vascular endothelial growth factors, Cardiovasc. Res. 65 (2005) 550–563. [7] B.K. McColl, M.E. Baldwin, S. Roufail, C. Freeman, R.L. Moritz, R.J. Simpson, K. Alitalo, S.A. Stacker, M.G. Achen, Plasmin activates the lymphangiogenic growth factors VEGF-C and VEGF-D, J. Exp. Med. 198 (2003) 863–868. [8] M.J. Karkkainen, P. Haiko, K. Sainio, J. Partanen, J. Taipale, T.V. Petrova, M. Jeltsch, D.G. Jackson, M. Talikka, H. Rauvala, C. Betsholtz, K. Alitalo, Vascular endothelial growth factor C is required for sprouting of the first lymphatic vessels from embryonic veins, Nat. Immunol. 5 (2004) 74–80. [9] M.E. Baldwin, M.M. Halford, S. Roufail, R.A. Williams, M.L. Hibbs, D. Grail, H. Kubo, S.A. Stacker, M.G. Achen, Vascular endothelial growth factor D is dispensable for development of the lymphatic system, Mol. Cell. Biol. 25 (2005) 2441–2449. [10] M.G. Achen, S.A. Stacker, Tumor lymphangiogenesis and metastatic spreadNew players begin to emerge, Int. J. Cancer 119 (2006) 1755–1760. [11] I. Van der Auwera, S.J. Van Laere, G.G. Van den Eynden, I. Benoy, P. van Dam, C.G. Colpaert, S.B. Fox, H. Turley, A.L. Harris, E.A. Van Marck, P.B.

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